Brown adipocyte progenitors in human skeletal muscle

ABSTRACT

This invention relates to brown adipose tissue (BAT) progenitor cells and methods for isolating BAT progenitor cells from skeletal muscle. BAT progenitor cell surface markers and medium and agents for inducing cell differentiation into brown adipocytes are also provided. In some embodiments, the BAT progenitor cell expresses a first cell surface marker associated with endothelial cells, the first cell surface marker being detectable in an antibody based assay using a first antibody. In addition, the BAT progenitor cell can be substantially free of a second cell surface marker associated with endothelial cells, the second cell surface marker being substantially undetectable in said antibody based assay using a second antibody. The BAT progenitor cell can also be substantially free of additional cell surface markers.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation patent application of U.S. patentapplication Ser. No. 16/409,926, filed May 13, 2019, now U.S. Pat. No.11,299,710, which is a divisional patent application of U.S. patentapplication Ser. No. 14/432,028, filed Mar. 27, 2015, now U.S. Pat. No.10,301,595, which is a U.S. national phase application of PCTInternational Patent Application No. PCT/US2012/064389, filed on Nov. 9,2012, which claims priority to and the benefit of U.S. ProvisionalPatent Application No. 61/558,152, filed on Nov. 10, 2011, each of whichis incorporated herein by reference in their entireties.

TECHNICAL FIELD

This invention relates to brown adipose tissue (BAT) progenitor cellsand methods for isolating BAT progenitor cells from skeletal muscle. Theinvention also relates to BAT progenitor cell surface markers and mediumand agents for inducing cell differentiation. The invention is usefulfor the study, prevention and treatment of various metabolic diseasessuch as obesity, type 2 diabetes, insulin-resistance and dyslipidemia.

BACKGROUND

The epidemic of obesity is closely associated with increases in theprevalence of diabetes, hypertension, coronary heart diseases, cancerand other disorders. The role of white adipose tissue is to storelipids, and it is associated with obesity. The role of the brown adiposetissue (“BAT”) is effectively the opposite. It is specialized in lipidcombustion and the dissipation of energy as heat. Indeed, the brownadipocyte contains lots of mitochondria (in which cellular combustionoccurs) and uniquely expresses BAT uncoupling protein-1 (“UCP1”). UCP1acts as an uncoupler of oxidative phosphorylation, resulting indissipation of energy as heat. The sympathetic nervous system stimulatesmitochondriogenesis and UCP1 expression and activity. BAT-associatedthermogenesis in rodents is increased upon exposure to low temperature(e.g., preventing hypothermia) or as a result of overeating, burningexcess absorbed fat and preventing weight gain. BAT, by modifyingsusceptibility to weight gain and by consuming large amounts of glucose,also improves insulin sensitivity. It therefore plays an important rolein the maintenance of body temperature, energy balance and glucosemetabolism.

Experiments with transgenic animals support the potential anti-obesityproperties of BAT. For example, the genetic ablation of BAT has beenreported to cause obesity, while genetic increase in the amount and/orfunction of BAT (and/or UCP1 expression) reportedly promotes a lean andhealthy phenotype. Specifically, mice with a higher amount of BAT gainless weight and are more insulin-sensitive than control mice. Recently,ectopic BAT depots were evidenced in the mouse muscle, which wereproposed to provide a genetic-based mechanism of protection from weightgain and metabolic syndrome.

Although UCP1 is reported to play a role in the control of energybalance in rodents and UCP1-expressing BAT is present in human neonates,it has long been thought that there was no physiologically relevant UCP1expression in adult humans. Indeed, UCP1-expressing BAT was thought todisappear early in life, and adult humans were thought to be devoid ofBAT.

As such, a need exists to carefully identify and study ways to providemore BAT in the adult body and/or stimulate UCP1 expression, for thestudy, prevention and treatment of various metabolic diseases such asobesity, type 2 diabetes, insulin-resistance dyslipidemia and type 1diabetes.

SUMMARY

Applicants have for the first time identified the presence of cells invarious tissues that are capable of differentiating into brownadipocytes. In one aspect, Applicants have identified a population ofsuch cells, which Applicants refer to as BAT progenitor cells, inskeletal muscle. The present disclosure provides methods for sortingcells from various tissues to identify and isolate BAT progenitor cells.In some embodiments, BAT progenitor cells are isolated from humanskeletal muscle. Methods are provided for differentiating BAT progenitorcells in vitro and in vivo into brown adipocytes. In some embodiments,BAT progenitor cells can be caused to differentiate in vivo into brownadipocytes in a human subject. The present invention also relates to BATprogenitor cell surface markers (e.g., CD31 and CD34) and medium andagents for inducing cell differentiation (e.g., PPARγ agonist and BMP7).

In some embodiments, BAT progenitor cells of the present disclosure canbe expanded in culture. In another aspect, differentiated BAT progenitorcell UCP1 mRNA expression is increased by agents such as cell-permeatingcAMP derivatives, peroxisome-proliferator-activated receptor (PPAR suchas PPARγ) agonists, and the like. BAT progenitor cells that have beendifferentiated into brown adipocytes may, in some embodiments, containlarge amounts of mitochondrial transcription factor A (mtTFA) and PPARγcoactivator-1α (PGC-1α), which are both involved in the control ofmitochondriogenesis, as well as of mitochondrial marker cytochromeoxidase IV (COX IV). Differentiated BAT progenitor cells can exhibit oneor more of the following characteristics: high levels of UCP1expression, high levels of uncoupled respiration, high respiration rate,high metabolic rate. Applicants provide differentiated cells that areequipped to metabolize glucose, oxidize fatty acids, and dissipateenergy as heat via uncoupling of oxidative phosphorylation.

The present disclosure provides methods for detection of UCP1 mRNA inthe skeletal muscle of adult humans, and methods for increasing itsexpression in vivo. Although prior studies concerning UCP1 expression inadult humans have focused on white adipose tissue, applicants disclosethe existence in, and isolation from, human skeletal muscle of brownadipose progenitor cells with a substantial potential for UCP1expression. In some embodiments, this reservoir of BAT progenitor cellscan be utilized for modulation of energy dissipation and for treatingobesity, diabetes, and metabolic diseases.

In some aspects, this disclosure provides methods for the identificationof BAT progenitor cells in human skeletal muscle and methods to isolatethese cells from human skeletal muscle samples. Also provided areconditions and agents (e.g., compounds, proteins, biologicals, and thelike) that promote the differentiation of these progenitor cells tobrown adipocytes in vitro, in vivo, or both. Methods are provided forusing these conditions and agents to treat metabolic diseases such asobesity, type 2 diabetes, insulin-resistance, dyslipidemia, and thelike.

The present disclosure provides assays that allow identification ofagents (e.g., compounds, proteins, biologicals, and the like) thatinduce the expression of the UCP1 gene, promote the differentiation ofBAT progenitor cells into brown adipocytes in vitro, promote thedifferentiation of BAT progenitor cells to brown adipocytes in vivo, orcombinations of these activities. According to some embodiments, agentsidentified in this manner can be used to treat metabolic diseases suchas obesity, type 2 diabetes, insulin-resistance, dyslipidemia, and thelike.

In one aspect, a brown adipose tissue (BAT) progenitor cell is provided.The BAT progenitor cell is isolated from human skeletal muscle andcapable of differentiating into brown adipocyte. The BAT progenitor cellexpresses a first cell surface marker associated with endothelial cells,said first cell surface marker being detectable in an antibody basedassay using a first antibody. In addition, the BAT progenitor cell issubstantially free of a second cell surface marker associated withendothelial cells, said second cell surface marker being substantiallyundetectable in said antibody based assay using a second antibody. Incertain embodiments, the BAT progenitor cell can be substantially freeof at least one of a third, fourth, and fifth cell surface marker. Forexample, the BAT progenitor cell can be substantially free of a thirdcell surface marker associated with hematopoietic cells, said third cellsurface marker being substantially undetectable in said antibody basedassay using a third antibody. The BAT progenitor cell can besubstantially free of the third cell surface marker and/or a fourth cellsurface marker associated with myogenic cells, said fourth cell surfacemarker being substantially undetectable in said antibody based assayusing a fourth antibody. The BAT progenitor cell may be substantiallyfree of the third and/or fourth cell surface marker, and/or a fifth cellsurface marker associated with pericytes, said fifth cell surface markerbeing substantially undetectable in said antibody based assay using afifth antibody.

In a further aspect, the present invention features a population ofcells isolated from skeletal muscle, comprising at least one BATprogenitor cell as described herein.

In another aspect, a method for identifying a BAT progenitor cell isprovided. The method includes: providing a population of cells isolatedfrom skeletal muscle; contacting the population of cells with a firstand a second antibody that are specific to a first and a second cellsurface marker, respectively, wherein the first and second cell surfacemarkers are each associated with endothelial cells; and determining, inan antibody based assay, a BAT progenitor cell that expresses the firstcell surface marker and is substantially free of the second cell surfacemarker. In some embodiments, the method can further include at least oneof: contacting the population of cells with a third antibody specific toa third cell surface marker associated with hematopoietic cells, saidthird cell surface marker being substantially undetectable in saidantibody based assay using the third antibody; contacting the populationof cells with a fourth antibody specific to a fourth cell surface markerassociated with myogenic cells, said fourth cell surface marker beingsubstantially undetectable in said antibody based assay using the fourthantibody; and/or contacting the population of cells with a fifthantibody specific to a fifth cell surface marker associated withpericytes, said fifth cell surface marker being substantiallyundetectable in said antibody based assay using the fifth antibody. Incertain embodiments, the method can further include separating thepopulation of cells by selecting cells that express the first cellsurface marker and that do not express the second cell surface markers.In some embodiments, the method can include separating the population ofcells by selecting cells that express the first cell surface marker andthat do not express the second, and at least one of the third, fourthand fifth cell surface markers. For example, said selecting can includeselecting cells that express CD34 and that do not express CD31, CD45,CD56 or CD146. The method can also include isolating the BAT progenitorcell and/or culturing the BAT progenitor cell in a proliferation medium,thereby expanding BAT progenitor cells ex vivo.

In yet another aspect, a method for inducing differentiation of BATprogenitor cells into brown adipocytes is provided. The method includes:providing the BAT progenitor cell described herein; exposing the BATprogenitor cell to a differentiation medium; and culturing the BATprogenitor cell in the differentiation medium to induce the BATprogenitor cell to differentiate into a brown adipocyte.

In still another aspect, the present invention features a method fortreating a metabolic disease or condition in a patient. The methodincludes: obtaining BAT progenitor cells from skeletal muscle of theindividual or a donor; propagating the BAT progenitor cells by culturingin a medium; and transplanting the cultured cells into the individual.In certain embodiments, the method can further include inducingdifferentiation of the BAT progenitor cells into brown adipocytes via exvivo culturing in a differentiation medium. In various embodiments, themetabolic disease is obesity, overweight, impaired glucose tolerance,insulin-resistance, type 2 diabetes, dyslipidemia, hypertension,cardiovascular disease, metabolic syndrome, Prader-Willi Syndrome, ortype 1 diabetes.

In a further aspect, a method for identifying an agent that inducesdifferentiation of a BAT progenitor cell into a brown adipocyte isprovided. The method includes: providing the BAT progenitor celldescribed herein; contacting the BAT progenitor cell with an agent;determining if the BAT progenitor cell exhibits an indicator ofdifferentiation into a brown adipocyte. In some embodiments, theindicator of differentiation can be an increase in one or more of thefollowing: expression of UCP1 protein or mRNA, expression of FABP4 (aP2)protein or mRNA, expression of PPARγ2 protein or mRNA, expression ofmtTFA protein or mRNA, expression of PGC-1α protein or mRNA, uncoupledrespiration, respiration rate, metabolic rate, glucose utilization rate,fatty acid oxidation rate, and a combination thereof.

In still a further aspect, a method for identifying an agent thatinduces expression or activity levels of UCP1 is provided. The methodincludes: providing the BAT progenitor cell described herein; contactingthe BAT progenitor cell with an agent; determining if the BAT progenitorcell exhibits an increase in UCP1 expression or activity.

The present invention also includes use of an agent identified using anyone of the methods described herein, in the manufacture of a medicamentfor the treatment of a metabolic disease, and/or for the treatment orprevention of hypothermia in a patient.

In some embodiments, the first cell surface marker can be CD34 and thefirst antibody can be an anti-CD34 antibody. In one embodiment, thesecond cell surface marker can be CD31 and the second antibody can be ananti-CD31 antibody. The third cell surface marker, for example, can beCD45 and the third antibody can be an anti-CD45 antibody. The fourthcell surface marker, for example, can be CD56 and the fourth antibodycan be an anti-CD56 antibody. The fifth cell surface marker, forexample, can be CD146 and the fifth antibody can be an anti-CD146antibody.

In various embodiments, the BAT progenitor cell can differentiate intothe brown adipocyte in a differentiation medium. For example, thedifferentiation medium can be Minimal Differentiation Medium (MDM). Thedifferentiation medium can further comprise a peroxisomeproliferator-activated receptor gamma (PPARγ) agonist and/or bonemorphogenic protein-7 (BMP7). In some embodiments, the BMP7, prior tothe addition of the differentiation medium, can be present in aproliferation medium which allows proliferation of the BAT progenitorcell.

Additional aspects of the present invention include isolated BATprogenitor cells, primary culture thereof, established or immortalizedcell line therefrom, cultured BAT progenitor cells or cell lines,transfected or transducted or infected or transformed cells or celllines, and any cells or cultures that may differentiate from such BATprogenitor cells or cell lines or transformants. These and otherfeatures of the present disclosure are set forth herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show immunohistochemical description (FIG. 1B) and FACSanalysis and sorting (FIG. 1A) of stroma-vascular cells in human fetalmuscle.

FIGS. 2A-2F show culture under adipogenic conditions of cells sortedfrom human fetal muscle and RT-PCR and Western blot analysis for theCD34+ cells. FIGS. 2A, 2B, 2C, 2D: Phase contrast; scale bar: 50 μm.FIG. 2E shows quantitative RT-PCR determination of UCP1 (empty columns)and leptin (gray columns) mRNA expression in CD34+ cells in primaryculture (PC) or expanded up to passage 3 (P3). FIG. 2F showsimmunohistochemical determination of UCP1 levels.

FIGS. 3A-3C show uncoupling of mitochondrial respiration and control ofUCP1 mRNA expression in human fetal muscle CD34+ cells. FIG. 3A showsuncoupling of mitochondrial respiration in isolated fetal muscle CD34+cells and in human adult white adipocytes grown in primary culture andfreshly trypsinized. FIG. 3B: The effects of cAMP derivatives on UCP1mRNA expression in CD34+ cells in PC or expanded up to P3. FIG. 3C: Theeffects of rosiglitazone (Rosi) 1 μM on UCP1 mRNA expression in CD34+cell PC or P3.

FIGS. 4A-4C show characterization of adult muscle and WAT cells inadipogenic culture.

FIGS. 4A, 4C: Phase contrast; scale bar: 50 μm.

FIG. 5 shows effects of rosiglitazone on UCP1 mRNA expression in humanskeletal muscle.

FIGS. 6A-6B show CD31− cell population before expansion (FIG. 6A:EMC309, passage 2;

FIG. 6B: EMC314, passage 1).

FIGS. 7A-7B show CD31+ cell population before expansion (FIG. 7A:EMC309, passage 4;

FIG. 7B: EMC314, passage 3).

FIGS. 8A-8B show CD56+ cell population before expansion (FIG. 8A:EMC309, passage 1;

FIG. 8B: EMC314, passage 3).

FIGS. 9A-9H show that CD31− cell population differentiate into brownadipocytes (FIGS. 9A-9D: EMC309, passage 4 at d14; FIGS. 9E-9H: EMC314,passage 3 at d14).

FIGS. 10A-10H show that CD31⁺ cells do not differentiate into adipocytes(FIGS. 10A-10D: EMC309, passage 6 at d14; FIGS. 10E-10H: EMC314, passage5 at d14).

FIGS. 11A-11H show that CD56⁺ cells do not differentiate into adipocytes(FIGS. 11A-11D: EMC309, passage 6 at d14; FIGS. 11E-11H: EMC314, passage5 at d14).

FIGS. 12A-12B show that significantly less RNA (reflecting a lowernumber of cells) was recovered from the CD31+ and CD56+ cells than fromthe CD31− cells grown in adipogenic differentiation medium (MDM),irrespective of the presence or absence of rosiglitazone or BMP7(+/−cmpd). Compare dark bars (MDM+/−cmpd) in CD31− samples vs. CD31+ andCD56+ samples. FIG. 12A: CD31-, CD31+, CD56+ cells. FIG. 12B: EMC309/314cells.

FIG. 13 shows that CD31− cells differentiate into adipocytes and expressUCP1. Exposure of CD31− cells to rosiglitazone or BMP7 robustlyincreases the differentiation of the cells and expression of UCP1.

FIG. 14 shows that cAMP and β-AR agonists have synergistic effects withPPARγ agonist on UCP1 mRNA expression. RDM=Minimal DifferentiationMedium+Rosiglitazone, iso=(−)-Isoproterenol.

FIG. 15 shows that differentiated brown adipocytes have significantlyincreased levels of PPARγ2 mRNA as compared to undifferentiated BATprogenitor cells. RDM=Minimal Differentiation Medium+Rosiglitazone,iso=(−)-Isoproterenol.

FIG. 16 shows respiration of the CD31− cells. The CD31− cellsdifferentiated into brown adipocytes (gray bars) have higher levels ofmitochondrial uncoupling, or proton leak (State 4 respiration), as wellas maximal (uncoupled) respiration (State 3u) vs. non-differentiatedcells (EGM2, white bars).

FIGS. 17A-17B show the quantification of UCP1 in CD31− cells byfluorescence immunohistochemistry. Differentiation of CD31− cells intobrown adipocytes without (FIG. 17A) or with (FIG. 17B) rosiglitazone.

FIGS. 18A-18B show the quantification of UCP1 (FIG. 18A, black bars),FABP4 (FIG. 18B, gray bars) and cyclophilin A mRNA by multiplexedreal-time PCR in undifferentiated (EGM2) and differentiated(rosiglitazone or BMP7) CD31− cells.

DETAILED DESCRIPTION

The present disclosure provides methods for identifying and isolatingBAT progenitor cells in and from various tissues, including, in someembodiments, the identification of common brown adipocyte progenitorcells in human skeletal muscle and isolation of such cells from humanskeletal muscle samples. In some embodiments, the cell sorting can bedone by immunohistochemical analysis of cell surface markers such as oneor more of cluster of differentiation/designation (“CD”) molecules CD31,CD34, CD45, CD56, and CD146. CD31 and CD34 can be used to identifyendothelial cells. Hematopoietic cells and myogenic progenitors can besorted based on identification of CD45 and CD56, respectively, on theircell surfaces. CD146 can be used to identify pericytes. In one aspect,expression of CD34 identifies a cell as a progenitor of a brownadipocyte. In a further aspect, a subpopulation of CD34+ cells representthe most pure human brown adipocyte progenitors known to date.

Flow cytometry, fluorescent-activated cell sorting (“FACS”), and othercell sorting techniques known in the art can be used for sorting cellsobtained from various tissues and for separating BAT progenitor cellsfrom other cells. Among other techniques known in the art, multi-colorFACS can be used to identify CD31-CD34+ endothelial cells. Additionally,one or more of CD146+ pericytes, CD45+ hematopoietic cells and/or CD56+myogenic progenitors can be separated and removed. Reverse transcriptasepolymerase chain reaction (“RT-PCR”) analysis can be used to confirm theabsence of hematopoietic cells and myogenic progenitors from thepopulations of CD34+ and CD146+ cells.

Applicants have unexpectedly found that a population of progenitors ispresent in skeletal muscle, and that this population is, in someembodiments, found in skeletal muscle but not in white adipose tissueand, in some embodiments, exclusively found in skeletal muscle (i.e.,not in other tissues). The skeletal muscle may be that of a human or ofany animal, and populations of progenitor cells may be diffuse in theskeletal muscle or concentrated in discrete regions. BAT progenitorcells may, in some embodiments, be found between myofibers. Skeletalmuscle BAT progenitor cells may be a stationary population or may bemobile both within skeletal muscle or other tissue and between and amongdifferent tissues. Further, BAT progenitor cells can be found in fetal,juvenile, and adult skeletal muscle.

The present teachings provide BAT progenitor cells isolated from varioustissues. For example, BAT progenitor cells isolated from human skeletalmuscle are provided. In some embodiments, the BAT progenitor cells arefound in skeletal muscle but not in white adipose tissue, and/or areexclusively found in skeletal muscle. Some BAT progenitor cells mayexpress UCP1, mitochondrial transcription factor A (mtTFA), and/or PPARγcoactivator-la (PGC-1α) as well as one or more of the correspondingmRNAs. The present disclosure provides methods for detection of BATprogenitor cells and/or UCP1 mRNA in the skeletal muscle of adulthumans. Although prior studies concerning UCP1 expression in adulthumans have focused on white adipose tissue, applicants disclose theexistence in, and isolation from, human skeletal muscle of brown adiposeprogenitor cells with a high potential for UCP1 expression. In someembodiments, a reservoir of BAT progenitor cells in skeletal muscleprovides a mechanism for modulating energy dissipation for treatment ofmetabolic diseases such as obesity, diabetes, and the like.

At least a portion of the population of progenitor cells present inskeletal muscle is capable of differentiating into genuine brownadipocytes, and, in some embodiments, a portion of the population ofprogenitor cells present in skeletal muscle are capable of beingdifferentiated in vitro into genuine brown adipocytes. The presentdisclosure provides methods for expanding BAT progenitor cell culturesand methods for differentiating BAT progenitor cells into genuine BATcells, including methods for differentiating previously sorted cells inan adipogenic medium. In some embodiments, differentiation of sortedprogenitor cells into brown adipocytes can be performed using conditionsthat sustain white adipocyte differentiation or by use of agentsdetermined to promote differentiation of progenitors into brownadipocytes.

Some embodiments utilize the presence of UCP1, FABP4 (aP2), PPARγ2,mitochondrial transcription factor A (mtTFA), and/or PPARγcoactivator-1α (PGC-1α) as well as one or more of the correspondingmRNAs, to identify BAT progenitor cells that have begun to at leastpartially differentiate. High metabolic rate or high levels of uncoupledrespiration, glucose utilization, fatty acid oxidation, or combinationsof the foregoing characteristics with each other or othercharacteristics, can be used to identify BAT progenitor cells that havebegun to at least partially differentiate. For purposes of thisdisclosure, BAT progenitor cells that have begun to at least partiallydifferentiate into brown adipocytes are referred to as “differentiatedbrown adipocytes.”

As an example, cells determined to express the CD34 marker (i.e., CD34+cells) can be differentiated into brown adipocytes by culturing inDMEM-Ham's F-12 medium containing 0.86 μM insulin, 10 μg/ml transferrin,0.2 nM triiodothyronine, 1 μM rosiglitazone, 100 μM3-isobutyl-1-methylxanthine, 1 μM dexamethasone and 1%penicillin-streptomycin. Other agents may also be used to promotedifferentiation of progenitor cells into brown adipocytes. In someembodiments, agents identified according to the teachings of thisdisclosure are used to promote differentiation of progenitor cells intobrown adipocytes. In some embodiments, differentiated brown adipocytesexhibit high levels of UCP1 expression, high levels of uncoupledrespiration, and/or high metabolic rate.

In another example, cells expressing the CD34 marker but aresubstantially free of the CD31 marker (i.e., CD34+CD31− cells) canrepresent a purer population of BAT progenitor cells than CD34+ cells(i.e., including both CD34+CD31− cells and CD34+CD31+ cells). In certainembodiments, an adipogenic differentiation medium not containing a PPARγagonist (e.g., rosiglitazone), called Minimal Differentiation Medium(MDM), was shown to be sufficient to induce the differentiation of atleast a proportion of adipocyte progenitor cells. The composition of MDMis: DMEM/Ham's F-12 50/50 Mix (3.151 g/1, 17.5 mM D-glucose, 3.651 g/1L-glutamine) (Cellgro #10-090-CV), 5 μg/ml (0.86 μM) insulin, 10 μg/mltransferrin, 0.2 nM 3,3′,5-triiodo-L-thyronine, 100 μM3-isobutyl-1-methylxanthine, 1 μM dexamethasone, 1%penicillin-streptomycin.

The present disclosure provides methods for increasing UCP1 mRNAexpression in BAT progenitor cells, differentiated brown adipocytes, orboth. For example, agents such as cell-permeating cAMP derivatives andperoxisome-proliferator-activated receptor-γ (PPARγ) agonists can beused to increase UCP1 mRNA expression in BAT progenitor cells,differentiated brown adipocytes, or both. Enhanced UCP1 expression canbe determined by methods known in the art, including measurement of UCP1mRNA by quantitative RT-PCR. Exemplary primers for use in RT-PCRanalysis of UCP1 mRNA are provided as SEQ ID NOS: 1-4 and 11-12.

BAT progenitor cells exposed to adipogenic media can contain higherlevels of UCP1 mRNA than BAT progenitor cells that are not exposed toadipogenic media. Cyclophilin mRNA levels can serve as a normalizingvalue (reflecting the number of cells or the total amount of RNA) forevaluating the abundance of UCP1 mRNA in a cell. In some embodiments,UCP1 mRNA levels in BAT progenitor cells not exposed to adipogenic mediaare not detectable using RT-PCR while UCP1 mRNA levels in differentiatedbrown adipocytes is detectable and can be normalized to cyclophilin mRNAlevels. As a comparative measure of UCP1 expression, UCP1 mRNA levels indifferentiated brown adipocytes can be compared to UCP1 mRNA levels incultured mouse brown adipocytes. The present disclosure provides UCP1mRNA levels in differentiated brown adipocytes of about 25% of the UCP1mRNA levels in cultured mouse brown adipocytes, while in otherembodiments the UCP1 mRNA level is about 25±10% or from about 15% toabout 30% of the UCP1 mRNA levels in cultured mouse brown adipocytes.The present disclosure contemplates UCP1 mRNA levels in differentiatedbrown adipocytes in a range of from about 5% to about 100% of the UCP1mRNA levels in cultured mouse brown adipocytes. In some embodiments, theUCP1 mRNA levels can be in excess of 100% of the UCP1 mRNA levels incultured mouse brown adipocytes.

Differentiated brown adipocytes can contain significantly higher levelsof UCP1 mRNA than cells in same-species or same-individual adultskeletal muscle biopsies. In addition, the quantity of UCP1 protein in adifferentiated brown adipocyte can be approximately equal to thequantity of UCP1 protein in same-species or same-individual fetal BAT.The present disclosure contemplates UCP1 mRNA levels in humandifferentiated brown adipocytes being approximately equivalent to UCP1mRNA levels in human brown adipocytes in vivo. In some embodiments theUCP1 mRNA level in a human differentiated brown adipocyte can be in arange from about 1% to many times greater than UCP1 mRNA levels in humanbrown adipocytes in vivo.

The present disclosure provides methods for increasing UCP1 mRNA levelsin BAT progenitor cells, differentiated brown adipocytes, or both. Insome embodiments, the methods provide for selectively increasing UCP1mRNA levels in BAT progenitor cells, differentiated brown adipocytes, orboth. PPARγ agonists can stimulate UCP1 mRNA production in both skeletalmuscle and differentiated brown adipocytes. For example, in someembodiments, the PPARγ agonist rosiglitazone selectively stimulates UCP1mRNA production in skeletal muscle or in differentiated brownadipocytes. Cell-permeating cAMP derivatives can stimulate UCP1 mRNAproduction in both skeletal muscle and in differentiated brownadipocytes. For example, in some embodiments the cell-permeating cAMPderivative 8-bromo-cAMP selectively stimulates UCP1 mRNA production inskeletal muscle or in differentiated brown adipocytes while in someembodiments the cell-permeating cAMP derivative(4-chlorophenylthio)-cAMP selectively stimulates UCP1 mRNA production inskeletal muscle or in differentiated brown adipocytes. For example, insome embodiments the combination of both the PPARγ agonist rosiglitazoneand either the cell permeant cAMP analog dibutyryl-cAMP or the β-ARagonist (−)-isoproterenol can additionally selectively stimulate UCP1mRNA production in skeletal muscle or in differentiated brown adipocytes(FIG. 14).

Mitochondrial transcription factor A (“mtTFA”) andperoxisome-proliferator-activated receptor-γ coactivator-1α (“PGC-1α”)are involved in the control of mitochondriogenesis. Differentiated brownadipocytes can contain large amounts of mtTFA, PGC-1α, or both. Thepresent disclosure provides differentiated brown adipocytes havingsignificantly increased levels of mtTFA mRNA, PGC-1α mRNA, or both, ascompared to undifferentiated BAT progenitor cells. Mitochondrial markercytochrome oxidase IV (COX IV) is involved with the mitochondrialrespiratory chain. The present disclosure provides differentiated brownadipocytes having significantly increased levels of COX IV mRNA ascompared to undifferentiated BAT progenitor cells. Fatty acid bindingprotein-4 (“FABP4” or “aP2”) and peroxisome-proliferator-activatedreceptor-γ2 (“PPARγ2”) are genes expressed only in adipocytes (brown andwhite). Differentiated brown adipocytes can contain large amounts ofFABP4, PPARγ2, or both. The present disclosure provides differentiatedbrown adipocytes having significantly increased levels of FABP4 mRNA,PPARγ2 mRNA, or both, as compared to undifferentiated BAT progenitorcells. For example, FIG. 15 shows that upon differentiation induced byRDM, RDM+cAMP or RDM+iso, PPARγ2 mRNA level increased by 10.4 fold, 9.2fold, and 6.8 fold, respectively.

Differentiated brown adipocytes according to some embodiments have highlevels of uncoupled respiration and/or high metabolic rate. Uncoupledrespiration can occur when protons leak across the inner mitochondrialmembrane rather than passing through the adenosine triphosphate synthase(“ATP Synthase”) enzyme to drive production of adenosine triphosphate(“ATP”). The energy released by the proton movement in theelectrochemical proton gradient across the membrane is dissipated asheat, rather than in the process of making ATP. Uncoupled respirationcan be measured as a function of the portion of cellular respiration(e.g., oxygen consumption) that occurs independently of ATP formation byATP Synthase. For example, oxygen consumption in the electron transportchain of oxidative phosphorylation in the presence of oligomycin, whichblocks the function of ATP Synthase, provides a measure of uncoupledrespiration.

The present disclosure provides differentiated brown adipocytes havingsignificantly increased levels of uncoupled respiration as compared toundifferentiated BAT progenitor cells. In some embodiments, the presentdisclosure provides differentiated brown adipocytes having levels ofuncoupled respiration of about 50% of total respiration. Someembodiments exhibit uncoupled respiration at levels in a range of fromabout 20% to about 50% of total respiration. Using the level ofuncoupled respiration in adult white adipocytes as a standard forcomparison, some embodiments exhibit uncoupled respiration in a range offrom about 1.5 to about 3.5 times greater than in adult whiteadipocytes. In some embodiments, the level of uncoupled respiration isabout 2.5 times greater than in adult white adipocytes. The presentdisclosure provides, among other things, differentiated brown adipocytesthat are equipped to metabolize glucose, oxidize fatty acids, anddissipate energy as heat via uncoupling of oxidative phosphorylation.

The present disclosure provides conditions and agents (e.g., compounds,proteins, biologicals, and the like) that promote the differentiation ofBAT progenitor cells to brown adipocytes, both in vitro and in vivo. Insome embodiments, the differentiation-promoting agent is: a PPARγactivator, modulator, or inhibitor (e.g., rosiglitazone), a PPARαactivator or modulator (e.g., clofibrate, GW9578), a PPARδ activator ormodulator (e.g., GW501516 or GW0742), a dual PPARα and PPARδ activatoror modulator, a pan-PPAR (α, δ, γ) activator or modulator (e.g.,GW4148), a PDE1 inhibitor (e.g., vinpocetine or IBMX), a PDE3 inhibitor(e.g., siguazodan or IBMX), a PDE4 inhibitor (e.g., rolipram or IBMX), aPDE7 inhibitor (e.g., BMS 586353 or BRL 50481 or IBMX), prostaglandinJ2, 9alpha,11beta-prostaglandin F2, 9beta,11alpha-prostaglandin F2, apepdide derived from the Pituitary adenylate cyclase-activatingpolypeptide (ADCYAP1 or PACAP) gene (PACAP propeptide, PACAP-relatedpeptide, PACAP-38 or PACAP-27), dibutyryl-cGMP, 8-Bromo-cGMP, a NRIP1(RIP140) inhibitor, a PTEN inhibitor (e.g., potassium bisperoxo(bipyridine) oxovanadate or dipotassium bisperoxo(5-hydroxypyridine-2-carboxyl) oxovanadate), an al-adrenergic full orpartial agonist (e.g., phenylephrine or cirazoline), an α2-adrenergicantagonist (e.g., yohimbine), an RXRα activator or modulator (e.g., LGD1069 (Targretin) or 9-cis retinoic acid), a PGC-1α activator, a PGC-1βinhibitor or activator, adiponectin or an activator of adiponectinreceptor AdipoR1 and/or AdipoR2, an NOS inhibitor or activator (e.g.,1400 W, 2-Ethyl-2-thiopseudourea or NG-nitro-L-arginine methyl ester(L-NAME) or adenosine), a Rho kinase-ROCK inhibitor (e.g., fasudil,HA1077), BDNF, a monoamine oxidase (MAO) A inhibitor and/or a MAO Binhibitor (e.g., isocarboxazid, moclobemide, selegiline), an activatorof SRC, an inhibitor of EGFR (e.g., RG-14620, erlotinib orZD1839-gefinitib or Argos protein), an inhibitor of FAAH (e.g., URB597),an inhibitor of MAPK 1 (e.g., PD98059) or 2 (e.g., PD98059) or 4 or 5 or7 or 8 (e.g., PD98059), an inhibitor of CDK9 (e.g.,1,5,6,7-Tetrahydro-2-(4-pyridinyl)-4H-pyrrolo[3,2-c]pyridin-4-onehydrochloride), a TGR5 agonist (e.g., oleanolic acid), an AMPK activator(e.g., AICAR), BMP-7, an mTOR inhibitor (e.g., rapamycin), an adenylatecyclase activator (e.g., forskolin), formoterol, salbutamol, bupropion,REV-5901, 24(S)-Hydroxycholesterol, 1,25-Dihydroxyvitamin D3,24,25-Dihydroxyvitamin D3, Prostaglandin J2, 15d-Prostaglandin J2,9alpha,11beta-Prostaglandin F2, 9beta,11alpha-Prostaglandin F2, Meadacid (20:3 n-9), Docosahexaenoic acid (22:6 n-3), Docosatrienoic acid(22:3 n-3), Docosapentaenoic acid, Lysophosphatidic acid, Bongkrekicacid, 3-Bromo-7-nitroindazole, Pregnenolone 16a carbonitrile,Epibatidine, a COX-2 inhibitor (e.g., NS-398), or combinations of any ofthe foregoing.

In some embodiments, treatment of a subject, including a human subject,with rosiglitazone results in an increase in the production of UCP1 mRNAin the subject's skeletal muscle. Treatment of subjects withrosiglitazone can, in some embodiments, induce the appearance ordifferentiation of brown adipocytes in skeletal muscle, enhanceexpression of the UCP1 gene in existing brown adipocytes in skeletalmuscle, or both. For example, in some embodiments the appearance ordifferentiation of brown adipocytes in skeletal muscle can be induced ina subject suffering from a metabolic disease. The brown adipocytes canprovide a glucose sink with high mitochondrial and cellular respirationand fatty acid oxidation rates, dissipating energy as heat (uncoupledoxidative phosphorylation). The subject metabolic rate can be enhanced,and a decrease in body weight can be induced. Induction of theappearance or differentiation of brown adipocytes can also yieldimprovements in insulin sensitivity, blood glucose homeostasis andcardiovascular disease risk factors. Brown adipocytes may furthersecrete factors that contribute to increased insulin sensitivity andimprove blood glucose homeostasis or cardiovascular health.

The present disclosure also provides assays that allow theidentification of agents (e.g., compounds, proteins, biologicals, andthe like) that promote the differentiation of BAT progenitor cells intobrown adipocytes and/or induce the expression of the UCP1 gene in vitro,in vivo, or both. Such agents can be identified by screening compounds,proteins, biologicals, and the like. For example, in some embodimentsisolated CD34+ cells can be used to screen agents for the ability toinduce expression of the UCP1 gene and/or differentiation of the CD34+cells into brown adipocytes. Agents identified in this manner can beused for a variety of research, diagnostic and therapeutic purposes,including, for example, treatment of metabolic diseases such as obesity,type 2 diabetes, insulin-resistance, dyslipidemia, and the like. In someembodiments, an agent identified by an assay according to the presentdisclosure is optimized for improvement of its physico-chemical and/orpharmacokinetic properties.

Expression of UCP1, FABP4 (aP2), PPARγ2, mtTFA, PGC-1α, and/or COX IV inBAT progenitor cells in vitro and in vivo can be enhanced according tomethods provided in the present disclosure. In some embodiments,exposure to adipogenic media can be used to stimulate increasedexpression of UCP1, FABP4 (aP2), PPARγ2, mtTFA, PGC-1α, and/or COX IV inBAT progenitor cells. Agents such as a PPARγ activator, modulator orinhibitor (e.g., rosiglitazone), a PPARα activator or modulator (e.g.,clofibrate, GW9578), a PPARδ activator or modulator (e.g., GW501516 orGW0742), a dual PPARα and PPARδ activator or modulator, a pan-PPAR (α,δ, γ) activator or modulator (e.g., GW4148), a PDE1 inhibitor (e.g.,vinpocetine or IBMX), a PDE3 inhibitor (e.g., siguazodan or IBMX), aPDE4 inhibitor (e.g., rolipram or IBMX), a PDE7 inhibitor (e.g., BMS586353 or BRL 50481 or IBMX), prostaglandin J2,9alpha,11beta-prostaglandin F2, 9beta,11alpha-prostaglandin F2, apeptide derived from the Pituitary adenylate cyclase-activatingpolypeptide (ADCYAP1 or PACAP) gene (PACAP propeptide, PACAP-relatedpeptide, PACAP-38 or PACAP-27), a NRIP1 (RIP140) inhibitor, a PTENinhibitor (e.g., potassium bisperoxo (bipyridine) oxovanadate ordipotassium bisperoxo (5-hydroxypyridine-2-carboxyl) oxovanadate), anal-adrenergic full or partial agonist (e.g., phenylephrine orcirazoline), an α2-adrenergic antagonist (e.g., yohimbine), an RXRαactivator or modulator (e.g., LGD 1069 (Targretin) or 9-cis retinoicacid), a PGC-1α activator, a PGC-1β inhibitor or activator, adiponectinor an activator of adiponectin receptor AdipoR1 and/or AdipoR2, an NOSinhibitor or activator (e.g., 1400 W, 2-Ethyl-2-thiopseudourea orNG-nitro-L-arginine methyl ester (L-NAME) or adenosine), a Rhokinase-ROCK inhibitor (e.g., fasudil, HA1077), BDNF, a monoamine oxidase(MAO) A inhibitor and/or a MAO B inhibitor (e.g., isocarboxazid,moclobemide, selegiline), an activator of SRC, an inhibitor of EGFR(e.g., RG-14620, erlotinib or ZD1839-gefinitib or Argos protein), aninhibitor of FAAH (e.g., URB597), an inhibitor of MAPK 1 (e.g.,PD98059), or 2 (e.g., PD98059) or 4 or 5 or 7 or 8 (e.g., PD98059), aninhibitor of CDK9 (e.g.,1,5,6,7-Tetrahydro-2-(4-pyridinyl)-4H-pyrrolo[3,2-c]pyridin-4-onehydrochloride), a TGR5 agonist (e.g., oleanolic acid), an AMPK activator(e.g., AICAR), BMP-7, an mTOR inhibitor (e.g., rapamycin), an adenylatecyclase activator (e.g., forskolin), formoterol, salbutamol, bupropion,REV-5901, 24(S)-Hydroxycholesterol, 1,25-Dihydroxyvitamin D3,24,25-Dihydroxyvitamin D3, Prostaglandin J2, 15d-Prostaglandin J2,9alpha,11beta-Prostaglandin F2, 9beta,11alpha-Prostaglandin F2, Meadacid (20:3 n-9), Docosahexaenoic acid (22:6 n-3), Docosatrienoic acid(22:3 n-3), Docosapentaenoic acid, Lysophosphatidic acid, Bongkrekicacid, 3-Bromo-7-nitroindazole, Pregnenolone 16a carbonitrile,Epibatidine, a COX-2 inhibitor (e.g., NS-398) or combinations thereofcan also be used to stimulate increased expression of UCP1, FABP4 (aP2),PPARγ2, mtTFA, PGC-1α, and/or COX IV in BAT progenitor cells.

EXAMPLES

Aspects of the present teachings may be further understood in light ofthe following examples, which should not be construed as limiting thescope of the present teachings in any way.

Example 1: Sorting and Differentiation of Muscle Vascular Cells

In fetal skeletal muscle, CD34 and CD146 were found, byimmunohistochemistry, to be expressed at the surface of endothelialcells and pericytes, respectively, although CD34 was also expressed bycells scattered in the inter-myofibrillar space. CD146+ pericytes havebeen found to surround CD34+ endothelial cells. A similar distributionof CD34+ and CD146+ cells was observed in adult skeletal muscle.

Vascular cells from seven independent fetal muscles (16-24 weeks ofgestation) were sorted using multi-color fluorescence-activated cellsorting (FACS). Hematopoietic (CD45+) cells were first gated out, aswere myogenic progenitors (CD56+). Then, endothelial cells(CD34+/CD146−) and pericytes (CD34−/CD146+) were sorted. TheCD34+/CD146−/CD45−/CD56− are designated thereafter as CD34+ cells andthe CD34−/CD146+/CD45−/CD56− as CD146+ cells. FIG. 1A shows CD34+/CD146−and CD34-/CD146+ cell purification. Dissociated cells were stained withPE-anti-CD34, FITC-anti-CD146, PE-Cy7-anti-CD56 and APC-Cy7-anti-CD45antibodies and run on a FACS Aria cell sorter. Following exclusion ofCD45+ and CD56+ cells (left panels), cells inside the CD34+ or CD146+gates were isolated. The CD34+ cells amounted to 8+1% of the startingfetal muscle cell population.

FIG. 1B shows RT-PCR analysis on CD34+/CD146-/CD45-/CD56− (CD34),CD34-/CD146+/CD45-/CD56− (CD146) and total non-sorted cells. Actin mRNAwas measured as a control. The CD34+ cells were shown not to becontaminated by detectable CD45+ hematopoietic or CD56+ myogenic cells.

Sorted cells were grown 4-6 days in EGM2 medium and 8-12 days in theadipogenic medium described under Materials and Methods. Theseconditions sustain white adipocyte differentiation in WAT primarycultures. FIGS. 2A-2F show CD34+(FIG. 2A) and CD146+(FIG. 2B, FIG. 2C)cells in primary cultures (PC) and CD34+(FIG. 2D) cells expanded inculture up to passage 3 (P3). Virtually all sorted fetal muscle CD34+cells differentiated into adipocyte-like multilocular cells (FIGS. 2A,2D). It is noteworthy that in cell culture, the multilocular structureis shared by white and brown adipocytes. In contrast, fetal muscleCD146+ cells grew very slowly under the conditions described above. Theydid not reach cell confluence and displayed a pericyte-like appearancecharacterized by a large size, spread out shape and irregular borders(FIGS. 2B and 2C). Occasional multilocular cells could be detected (FIG.2C) The morphology of CD34+ cells expanded in culture for up to 3passages (4 weeks) under the conditions described above was similar tothat observed in primary culture, although the size of mature adipocyteswas smaller (FIG. 2D).

In certain embodiments, CD45+ cells and/or CD56+ cells can be left inthe CD34+/CD146-population; that is, sort CD34+/CD146− cells withoutfirst gating out at least one of CD45+ cells and CD56+ cells. It hasbeen observed that the CD45+ and CD56+ populations can be relativelysmall (e.g., each population less than 5%, less than 4%, less than 3%,less than 2% or less than 1% of the total stroma-vascular cells frommuscle). Therefore, a relatively pure brown adipocyte progenitorpopulation can be isolated without the need to gate out at least one ofCD45+ and CD56+ populations. Furthermore, CD45+ cells are hematopoieticprogenitors and do not grow or differentiate significantly in theadipogenic media. CD56+ cells are muscle precursors and do not grow ordifferentiate significantly in the adipogenic media. Thus, the presenceof CD45+ cells and/or CD56+ cells do not significantly affect theproliferation and/or differentiation of the CD34+ cells.

Example 2: UCP1 Expression in Cultivated CD34+ Cells

The remarkable adipocyte-like differentiation of fetal muscle CD34+cells was an incentive for further characterization. Strikingly,quantitative RT-PCR revealed a high level of UCP1 mRNA in these cells.FIG. 2E shows quantitative RT-PCR determination of UCP1 (empty columns)and leptin (gray columns) mRNA expression in CD34+ cells in primaryculture (PC) or expanded up to passage 3 (P3). The mean UCP1 mRNA levelnormalized to cyclophilin A was 1797±510 arbitrary units (i.e., ±s.e.m.of arbitrary values normalized using the corresponding cyclophilin Avalues; n=4-7), corresponding to a cycle threshold (Ct) of 22 for 25 ngof cDNA in the assay.

For comparison, the mean UCP1 mRNA level normalized to cyclophilin A inmouse brown adipocytes differentiated in culture was 7715±2649 (n=10)arbitrary units. Therefore, the level of UCP1 mRNA in human CD34+ cellsamounted to almost one fourth of that in mouse brown adipocytes inculture. Human fetal BAT was not used as a positive control forquantitative RT-PCR analysis because the risk of RNA degradation washigh due to the time elapsed after the termination of the pregnancy. Theamplicon was cloned and sequenced and found to be 100% identical tohuman UCP1. In fetal muscle CD34+ cells expanded up to passage 3 a highUCP1 mRNA expression, amounting to 43% of that detected in primarycultured cells, was still observed. UCP1 mRNA expression was notdetected in non-differentiated fetal muscle CD34+ cells or in CD146+cells in primary culture. The level of leptin mRNA was 9.9±5.5 and 71±52arbitrary units in primary cultured and expanded cells, respectively(FIG. 2E).

Example 3: Additional Phenotyping of the CD34+ Cells

To better characterize the gene expression pattern of the fetal muscleCD34+ cells expanded in culture a gene chip analysis was performed. Thelevels of expression of several representative gene mRNAs withsignificant Detection P-Values (p<0.01) are shown in Table 1 andcompared with those in human muscle biopsies. The following proteinmRNAs were chosen: UCP1 as a reference gene, mitochondrial transcriptionfactor A (mtTFA) and peroxisome-proliferator-activated receptor (PPARγ)and PPARγ coactivator-1α (PGC-1α), which are involved in the control ofthermogenesis and mitochondriogenesis, enzymes of the mitochondrialrespiratory chain succinate dehydrogenase (SDH) and cytochrome oxidaseIV (COX IV), enzymes of the fatty acid degradation pathway, carnitinepalmitoyltransferase IB (CPTIB), acyl-CoenzymeA dehydrogenases longchain (ACAD) and C-4 to C-12 straight chain (ACADM), and the skeletalmuscle markers myogenin, myogenic factor 5 (Myf5) and myogenicdifferentiation 1 (MyoD1). Cidea, which is highly expressed in BAT andmay act as a suppressor of UCP1 activity [16], was chosen as a BATmarker. The Genbank accession numbers of these genes are shown in thesupplemental data.

TABLE 1 Accession Human muscle mRNA No. CD34+ cells biopsies UCP1NM_021833 94 n.s. mTFA NM_003201.1 413 205 PPARγ NM_138712.2 3326 84PGC-1α NM_013261.2 137 619 COX IV NM_001861.2 13,082 13,407 SDHNM_003000.1 2390 5187 CPIB NM_004377.2 99 639 ACAD NM_032169.3 1032 141ACADM NM_000016.2 599 1640 Myogenin NM_002479.3 n.s. 267 Myf5 NM_05593n.s. 21 MyoD1 NM_002478 n.s. 12 Cidea NM_198289.1 337 n.s.

The data in Table 1 are expressed as the average Illumina signal. TheDetection P-Values are <0.01. The following abbreviations are used:n.s., not significant; mtTFA, mitochondrial transcription factor A;PPARγ, peroxisome-proliferator-activated receptor-γ; PGC-1α, PPARγcoactivator-la; COX IV, cytochrome oxidase IV; SDH, succinatedehydrogenase; CPT1B, carnitine palmitoyltransferase IB; ACAD,acyl-CoenzymeA dehydrogenases long chain; ACADM, C-4 to C-12 straightchain; Myf5, myogenic factor 5; MyoD1, myogenic differentiation 1.

UCP1 was significantly expressed in fetal muscle-expanded CD34+ cellsbut not in adult muscle biopsies (for which p=0.12). The levels of mRNAexpression of the selected genes in expanded CD34+ cells from fetalmuscle were comparable with those of the adult muscle biopsies with theexceptions of PGC-1α and CPT1B mRNAs (which were about 5-fold lessexpressed in the cells) and of the PPARγ and ACAD mRNAs (which were 40-and 7-fold less expressed, respectively in the muscle biopsies). Themuscle markers myogenin, Myf5 and MyoD1 mRNA were significantlyexpressed in the muscle but not in the cells whereas the BAT markerCidea mRNA was expressed in the cells but not in the muscle. Noβ3-adrenoceptor mRNA could be detected in the gene chip analysis. It isnoteworthy, however, that β3-adrenoceptor mRNA was detected byquantitative RT-PCR (arbitrary value 0.084±0.044 with cyclophilin A as areference; n=4) in fetal muscle CD34+ cells in primary culture.Measurements of mtTFA, PGC-1α and COX IV were also performed byquantitative RT-PCR to confirm the gene chip data with a differenttechnique. The results were confirmatory, showing that fetal muscleCD34+ cells in primary culture express high levels of mtTFA, PGC-1α andCOX IV mRNA [amounting to 306±117, 385±294, and 23,400±10,300 arbitraryunits (n=3-4), respectively], using cyclophilin A as a reference.

The UCP1 protein, as assessed by Western blotting with an anti-mouseantibody cross-reacting with human UCP1 (80% identity), was as abundantin primary cultured fetal muscle CD34+ cells as in fetal BAT. FIG. 2(F)shows representative Western blot analysis of UCP1 and glyceraldehydephosphate dehydrogenase (GAPDH) proteins in tissue or whole cellextracts. Interscapular BAT of a 19-week fetus (Lane 1), CD34+ cells inprimary culture (Lane 2), and skeletal muscle of an adult human (Lane 3)are shown. 25 μg of protein was loaded into each lane.

Example 4: Uncoupling of Oxidative Phosphorylation

To get insight into the possible function of UCP1 in muscle-derivedcells, mitochondrial respiration of isolated cultured human fetal muscleCD34+ cells and human adult white adipocytes was compared. Basalrespiration was defined as the antimycin A-sensitive oxygen consumption.Uncoupled respiration (proton leak) was defined as the percentage ofbasal respiration insensitive to the ATP synthase blocker oligomycin.

FIG. 3A shows uncoupling of mitochondrial respiration in isolated fetalmuscle CD34+ cells and in human adult white adipocytes grown in primaryculture and freshly trypsinized. The results are means s.e.m; *p<0.05.n=3. The ratios of uncoupled to total respiration were 47±12% and 19±2%in human fetal muscle CD34+ cells and adult white adipocytes,respectively.

FIG. 16 shows that the CD31− cells, upon differentiation into brownadipocytes, display more highly uncoupled respiration and greaterrespiration capacity compared to the same cells prior todifferentiation.

Example 5: Modulation of UCP1 Expression in Cultured CD34+ Cells

UCP1 mRNA expression in fetal muscle CD34+ cells could be modulated bydrug treatment. Cell-permeating cAMP derivatives strongly stimulated (7to 8-fold) UCP1 mRNA expression in both primary cultured and expandedcells. The effects of cAMP derivatives, 8-bromo-cAMP, 0.25 mM or(4-chlorophenylthio)-cAMP, 0.25 mM (cAMP) on UCP1 mRNA expression inCD34+ cells in primary culture (PC) or expanded up to passage 3 (P3) areshown in FIG. 3B. All the cells were grown for 4-6 days in EGM2 mediumand then placed for 8-12 days in the adipogenic medium described underMaterials and Methods. The results are means±s.e.m. of arbitrary valuesnormalized using the corresponding cyclophilin A values. They areexpressed in % of their respective untreated (control) values consideredas 100% (*p<0.05, n=3-6).

Rosiglitazone, a PPARγ agonist, had no effect in primary culture cellsbut strongly stimulated (8-fold) UCP1 mRNA expression in expanded cells.The effects of rosiglitazone (Rosi) 1 μM on UCP1 mRNA expression inCD34+ cell PC or P3 are shown in FIG. 3C. The results are expressed asin FIG. 3B (**p<0.01, n=4-7).

Example 6: Muscle Specificity and Persistence Throughout Life of HumanBrown Adipocyte Progenitors

The derivation of UCP1-expressing cells from human fetal muscle raisedthe question of the restriction of brown adipocyte progenitors to thistissue and to the fetal stage. To address this issue, CD34+ cellspurified by FACS from human fetal pancreas, lung and liver were culturedunder the same adipogenic conditions as fetal muscle CD34+ cells. Thesorted cells grew slowly and only a small proportion of them becamemultilocular. UCP1 mRNA was not expressed in pancreas or lung cells;however, a minor expression was measured in liver cells, which amountedto 2% of that detected in fetal muscle CD34+ cells (not shown).

CD34+ cells sorted from 4 adult (50-78 years) human skeletal musclesamples, grown in primary culture (PC) under adipogenic conditions, alsodifferentiated into multilocular cells. These cells were interspersedwith other types of cells, some of them containing small lipid droplets(FIG. 4A). The level of UCP1 mRNA (370±132 arbitrary units) was 21% ofthat detected in primary cultured fetal muscle CD34+ cells. In contrast,leptin expression (75±69 arbitrary units) was 7.6-fold higher than infetal cells. FIG. 4B shows quantitative RT-PCR determination of UCP1(empty column) and leptin (gray column) mRNA expression. All the cellswere grown for 4-6 days in EGM2 medium and then placed for 8-12 days inthe adipogenic medium described under Materials and Methods. The resultsare the mean±s.e.m. of arbitrary values normalized to the correspondingcyclophilin A values (n=4-5). CD34+ cells sorted from 4 adult (45-55years) human WAT samples were also grown in primary culture (PC) underadipogenic conditions. They became partially multilocular (FIG. 4C), butdid not express UCP1 mRNA.

Example 7: Detection of UCP1 mRNA Expression in Human Muscle and Effectof Rosiglitazone In Vivo

Brown adipocyte progenitors of adult human skeletal muscle candifferentiate in vivo and give rise to UCP1 expressing cells. Thepresence of UCP1 mRNA in the adult human skeletal muscle was trackedusing a high sensitivity RT-PCR technique and, in fact, low levels ofUCP1 mRNA were detected in the rectus abdominus muscle of 10 leansubjects (UCP1/cyclophilinA ratio: 24±9). The PCR-amplified fragment wassequenced and found to be 100% identical to human UCP1. The UCP1 mRNAlevel in adult human muscle was 75-fold lower than that in fetal muscleCD34+ cells in culture.

Since the PPARγ agonist rosiglitazone was a strong inducer of UCP1 mRNAexpression in muscle CD34+ cells in culture, the effect of this compoundin vivo in humans was investigated. Vastus lateralis muscle biopsiesfrom 7 obese patients with type 2 diabetes mellitus treated for themanagement of their metabolic syndrome with rosiglitazone were used. Thebiopsies were obtained before and after 8 weeks of treatment withrosiglitazone (2×4 mg per day). The treatment with rosiglitazoneresulted in a significant improvement of the patients' insulinresistance and diabetes. In that study rosiglitazone, concomitantly withthe improvement in insulin sensitivity, increased the level ofexpression of UCP1 in muscle by about 1.6-fold. FIG. 5 shows thequantitative RT-PCR determination of UCP1 mRNA expression. The resultsare the means±s.e.m. of arbitrary values normalized using thecorresponding cyclophilin A values (n=7, *p<0.05 vs. control). Since theRT-PCR conditions used were different, the arbitrary values of thisfigure do not provide a direct comparison to those of FIGS. 2A-4C.

In FIG. 5, showing UCP1 mRNA levels in skeletal muscle biopsies from apatient group (n=7), “control” corresponds to levels before treatment,and “Rosi” corresponds to levels after treatment (8 weeks) withrosiglitazone. Being a longitudinal study the effect of rosiglitazone onUCP1 levels in each individual (comparison of individual values for the“control”-before and “Rosi”-after conditions) were determined. Startingwith 25 ng cDNA (produced by reverse transcription of RNA) the thresholdof detection (Ct) during real-time PCR was about 22 for UCP1 and about18 for cyclophilin. The effect of rosiglitazone (UCP1 level at end oftreatment vs. before treatment) were as follows:

Patient 1: 50% increase (to 150%, control=437, Rosi=652 arbitrary units)Patient 2: no change (to 100%, control=444, Rosi=453 arbitrary units)Patient 3: 80% increase (to 180%, control=378, Rosi=677 arbitrary units)Patient 4: 180% increase (to 280%, control=260, Rosi=730 arbitraryunits)Patient 5: 8% increase (to 108%, control=553, Rosi=600 arbitrary units)Patient 6: 310% increase (to 410%, control=135, Rosi=556 arbitraryunits)Patient 7: 10% increase (to 110%, control=128, Rosi=142 arbitrary units)

Strong effects of rosiglitazone, varying between 1.5- and 4.1-fold, wereobserved in 4 out 7 patients. This result suggests that rosiglitazoneinduced the appearance of brown adipocytes and/or enhanced theexpression of the UCP1 gene in existing brown adipocytes in the skeletalmuscle of the patients. This effect of the PPARγ agonist may play a keyrole in the therapeutic effect of this agent as an insulin-sensitizer.

Example 8: Screening of Potential Modulators of the Human UCP1Promoter/Enhancer Region

The identified and isolated CD34+ cells can be used as a tool toidentify agents (compounds, proteins, biologicals, and the like) thatinduce the differentiation of these cells into brown adipocytes ormodulate the expression of UCP1. For example, an RT-PCR based approachcan be used to measure UCP1 mRNA levels which may be affected by certainagents. Alternatively, using a tagged UCP1 protein as marker,immunohistochemistry (e.g., fluorescence) can be used to detect changesin UCP1 protein level to screen for agents that modulate UCP1expression.

For this purpose and by way of example, a large region (6 kb) of DNAupstream (in 5′) of the transcription start site of the human UCP1 gene(containing the promoter/enhancer region) has been cloned into areporter/MAR GFP (Green Fluorescent Protein) or luciferase. Thisconstruct has been used to transfect CD34+ cells, and the cells grown inmultiwell plates and screened for agents that increase the fluorescence(GFP) or luminescence (luciferase) of the cells, reflecting induction ofgene expression (and thus increased UCP1 expression). This allows theidentification of agents that can enhance the differentiation of CD34+cells into brown adipocytes and/or the expression of UCP1 by enhancingthe transcription of the UCP1 gene and/or by enhancing the translationof the UCP1 transcript, and/or by stabilizing the UCP1 transcript orprotein.

For example, a PPARγ modulator or activator like rosiglitazone can beused to promote the differentiation of CD34+ progenitor cells into brownadipocytes (FIGS. 3C and 5). Another example is the use of cAMPderivatives like, 8-bromo-cAMP and/or (4-chlorophenylthio)-cAMP (FIG.3B) or protein kinase A (PKA) activators or phosphodiesteraseinhibitors. Another example is the use of triiodothyronine (T3), otherthyroid hormones, agonists or modulators of the thyroid hormonereceptors TRa and/or TRβ. Another example is to use β-adrenergicagonists like isoproterenol (pan-agonist) or specific β1-, β2-,β3-agonists or modulators, either alone or in combination with a PPARγmodulator or activator like rosiglitazone. Another is the use ofmodulators of the candidate receptors revealed by gene chip studies orof target genes in the signaling pathway downstream these receptors.

Example 9: Gene Chip Studies

Gene chip studies were performed to identify molecular pathways thatplay a role in the differentiation of CD34+ progenitor cells into brownadipocytes and/or the induction of the expression of UCP1. CD34+ cellswere isolated from human skeletal muscle biopsies, and were used in twostudies: (1) cAMP study: CD34+ cells were differentiated as described inMaterials and Methods (Control) plus addition of vehicle (Control 1sample) or cAMP (cAMP sample); and (2) Rosiglitazone study: CD34+ cellswere differentiated as described in Materials except that rosiglitazonewas omitted from the adipogenic medium (Control 2 sample). Rosiglitazonewas added only to the second sample (Rosiglitazone sample) in thisstudy. As discussed above, these compounds have been shown to promotethe differentiation of CD34+ cells into brown adipocytes and theexpression of UCP1 (see FIGS. 3B and 3C).

Total RNA was purified from these samples, and transcriptional profileswere assessed with Illumina Human WG-6 BeadChip (Expression Analysis,Inc., Durham, N.C.). Results were analyzed with Ingenuity PathwayAnalysis 7.0 (trial version). These results were used to determine whatmolecular pathways are involved in the differentiation of CD34+ cellsinto brown adipocytes, and, more importantly, what molecular targets canbe used for the development of agents that promote the appearance ofbrown adipocytes and the expression of UCP1.

This work showed that the following actions/agents should promote brownadipocyte development: a PPARγ activator, modulator or inhibitor (e.g.,rosiglitazone), a PPARα activator or modulator (e.g., clofibrate,GW9578), a PPARδ activator or modulator (e.g., GW501516 or GW0742), adual PPARα and PPARδ activator or modulator, a pan-PPAR (α, δ, γ)activator or modulator (e.g., GW4148), a PDE1 inhibitor (e.g.,vinpocetine or IBMX), a PDE3 inhibitor (e.g., siguazodan or IBMX), aPDE4 inhibitor (e.g., rolipram or IBMX), a PDE7 inhibitor (e.g., BMS586353 or BRL 50481 or IBMX), prostaglandin J2,9alpha,11beta-prostaglandin F2, 9beta,11alpha-prostaglandin F2, apeptide derived from the Pituitary adenylate cyclase-activatingpolypeptide (ADCYAP1 or PACAP) gene (PACAP propeptide, PACAP-relatedpeptide, PACAP-38 or PACAP-27), a NRIP1 (RIP140) inhibitor, a PTENinhibitor (e.g., potassium bisperoxo (bipyridine) oxovanadate ordipotassium bisperoxo (5-hydroxypyridine-2-carboxyl) oxovanadate), anal-adrenergic full or partial agonist (e.g., phenylephrine orcirazoline), an α2-adrenergic antagonist (e.g., yohimbine), an RXRαactivator or modulator (e.g., LGD 1069 (Targretin) or 9-cis retinoicacid), a PGC-1α activator, a PGC-1β inhibitor or activator, adiponectinor an activator of adiponectin receptor AdipoR1 and/or AdipoR2, an NOSinhibitor or activator (e.g., 1400 W, 2-Ethyl-2-thiopseudourea orNG-nitro-L-arginine methyl ester (L-NAME) or adenosine), a Rhokinase-ROCK inhibitor (e.g., fasudil, HA1077), BDNF, a monoamine oxidase(MAO) A inhibitor and/or a MAO B inhibitor (e.g., isocarboxazid,moclobemide, selegiline), an activator of SRC, an inhibitor of EGFR(e.g., RG-14620, erlotinib or ZD1839-gefinitib or Argos protein), aninhibitor of FAAH (e.g., URB597), an inhibitor of MAPK 1 (e.g.,PD98059), or 2 (e.g., PD98059) or 4 or 5 or 7 or 8 (e.g., PD98059), aninhibitor of CDK9 (e.g.,1,5,6,7-Tetrahydro-2-(4-pyridinyl)-4H-pyrrolo[3,2-c]pyridin-4-onehydrochloride), a TGR5 agonist (e.g., oleanolic acid), an AMPK activator(e.g., AICAR), BMP-7, an mTOR inhibitor (e.g., rapamycin), an adenylatecyclase activator (e.g., forskolin), formoterol, salbutamol, bupropion,REV-5901, 24(S)-Hydroxycholesterol, 1,25-Dihydroxyvitamin D3,24,25-Dihydroxyvitamin D3, Prostaglandin J2, 15d-Prostaglandin J2,9alpha,11beta-Prostaglandin F2, 9beta,11alpha-Prostaglandin F2, Meadacid (20:3 n-9), Docosahexaenoic acid (22:6 n-3), Docosatrienoic acid(22:3 n-3), Docosapentaenoic acid, Lysophosphatidic acid, Bongkrekicacid, 3-Bromo-7-nitroindazole, Pregnenolone 16a carbonitrile,Epibatidine, a COX-2 inhibitor (e.g., NS-398) or combinations of any ofthe foregoing.

Example 10: Sorting of Muscle Vascular Cells Using CD31 as an AdditionalMarker

With the aim of further purifying the CD34+ cells in search for thepurest physiological brown adipocyte progenitors, stroma-vascular cellswere purified from two independent samples of human fetal skeletalmuscle (18-19 weeks of gestation). Following the CD45, CD56, CD34, andCD146 sorting described above, an additional step of FACS sorting usingan anti-CD31 antibody was performed. The cells were sorted based on thepresence of the mature endothelial cell surface marker CD31 (CD31+) orabsence of that marker (CD31-). It was hypothesized that the CD31−subpopulation may be a purer progenitor of human brown adipocytes. TheCD31− cell fraction amounted to about 80% of the CD34+ cell populationderived from skeletal muscle. For purpose of simplicity,CD34+/CD45−/CD56−/CD146−/CD31− are called “CD31-” cells [EMC309 (CD31-)and EMC314 (CD31-)]. CD34+/CD45−/CD56−/CD146−/CD31+ are called “CD31+”cells [EMC309 (CD31+) and EMC314 (CD31+)]. In addition, the myogenicprogenitor cells expressing the cell surface antigen CD56 (CD56+) werealso sorted and cultured: CD34+/CD45−/CD56+(from EMC314) andCD45−/CD56+(from EMC309) are called “CD56+” cells [EMC309 (CD56+) andEMC314 (CD56+)].

The sorted cells (CD31-, CD31+, and CD56+ cell populations) were grownunder the same conditions as described above, i.e., 2-4 days inproliferation medium (EGM2) and 10-16 days in adipogenic differentiationmedium. Micrographs of the isolated cells in culture in EGM2 mediumbefore expansion are shown in FIGS. 6A-8B.

The capability of the CD31-, CD31+, and CD56+ cell populations todifferentiate into adipocytes was first tested in adipogenicdifferentiation medium in the absence of rosiglitazone. An adipogenicdifferentiation medium not containing a PPARγ agonist (e.g.,rosiglitazone), called Minimal Differentiation Medium (MDM), was shownto be sufficient to induce the differentiation of at least a proportionof adipocyte progenitor cells. The composition of MDM is: DMEM/Ham'sF-12 50/50 Mix (3.151 g/1, 17.5 mM D-glucose, 3.651 g/1 L-glutamine)(Cellgro #10-090-CV), 5 μg/ml (0.86 μM) insulin, 10 μg/ml transferrin,0.2 nM 3,3′,5-triiodo-L-thyronine, 100 μM 3-isobutyl-1-methylxanthine, 1μM dexamethasone, 1% penicillin-streptomycin.

An appreciable fraction (approximately 20%) of the CD31− cellsdifferentiated into adipocyte-like multilocular cells when exposed toMinimal Differentiation Medium (FIG. 9B for EMC309, FIG. 9F for EMC314),and virtually all the CD31− cells differentiated into adipocyte-likemultilocular cells when exposed to MDM containing a brownadipocyte-promoting compound such as the PPARγ activator rosiglitazoneor BMP7 [26-28] (FIG. 9C-D, 9G-H).

It was also found that the addition of a PPARγ agonist (e.g.,rosiglitazone at 1 microM) either from the onset of differentiation (day0) or, instead, for 2-3 days only starting 2 or 3 days before theinduction of differentiation (from day −3 or −2 to day 0) stronglypromoted the differentiation of the CD31− cells into brown adipocytes.Similarly, addition of bone morphopgenic protein-7 (BMP7 at 6 nM) beforethe induction of differentiation (from day −3 or −2 to day 0) stronglypromoted the differentiation of the CD31− cells into brown adipocytes.

In contrast, the CD31+ cells (FIGS. 10A-10H) and the CD56+(FIGS.11A-11H) cells did not grow well in MDM, and did not differentiate intoadipocyte-like multilocular cells under the conditions described above,with or without rosiglitazone or BMP7.

All cell populations (CD31-, CD31+ and CD56+) grew well in proliferationmedium (EGM2) over the whole 12-20 day study (FIGS. 9A and 9E for CD31-,FIGS. 10A and 10E for CD31+, FIGS. 11A and 11E for CD56+) but only theCD31− cells grew without detaching in MDM with or without rosiglitazoneor BMP7. The difference in cell density at day 14-16 between the CD31−and the CD31+ or CD56+ cells when grown in MDM is illustrated in themicrographs in FIGS. 9-11 (compare FIG. 9B, 9F to FIG. 10B, 10F or FIG.11B, 11F; FIG. 9C, 9G to FIG. 10C, 10G or FIG. 11C, 11G; FIG. 9D, 9H toFIG. 10D, 10H or FIG. 11D, 11H) as well as in the amount of cyclophilinmRNA (reflecting the number of cells) measured in the cultured cells atday 16 (FIG. 12A and FIG. 12B).

In certain embodiments, CD45+ cells and/or CD56+ cells can be left inthe CD31−/CD34+/CD146− population; that is, sort CD31-/CD34+/CD146−cells without first gating out at least one of CD45+ cells and CD56+cells. As discussed above, the CD45+ and CD56+ populations can berelatively small (e.g., each population less than 5%, less than 4%, lessthan 3%, less than 2% or less than 1% of the total stroma-vascular cellsfrom muscle), and do not grow or differentiate significantly in theadipogenic media. Therefore, a relatively pure brown adipocyteprogenitor population can be isolated without the need to gate out atleast one of CD45+ and CD56+ populations. Furthermore, the presence ofCD45+ cells and/or CD56+ cells do not significantly affect theproliferation and/or differentiation of the CD34+ cells.

Example 11: UCP1 Expression in Cultured CD31−, CD31+ and CD56+ Cells

Quantitative RT-PCR revealed a high level of UCP1 mRNA in thedifferentiated CD31− cells (normalized with cyclophilin A mRNA). Ascompared to the EGM2 control condition the levels of UCP1 expressionwere increased by 10-21-fold in MDM, 507-553-fold with rosiglitazone,and 78-127-fold with BMP7 (FIG. 13).

The proportion of differentiated (adipocyte) cells as well as theexpression of UCP1 was highly increased by addition of either: (a) 1microM rosiglitazone (a PPARγ agonist) to the MDM starting at day 0(MDM+rosi) (FIG. 13), or (b) 6 nM BMP7 (Bone Morphogenic Protein-7) onlyin the proliferation medium (EGM2) two or three days before induction ofdifferentiation, i.e., from day −2 or day −3 to day 0 (FIG. 13).

FIG. 13 shows that exposure of CD31− cells to rosiglitazone or BMP7robustly increases the differentiation of the cells and expression ofUCP1. UCP1 mRNA expression in human fetal muscle-derived CD31− cells atday 16 of differentiation were measured.

All the cells were grown for 3 days in proliferation medium (EGM2) andthen (on day 0) placed for 16 days in adipogenic differentiation medium(Minimal Differentiation Medium, MDM). The “EGM2” cells were maintainedin EGM2 medium all along. Rosiglitazone (1 microM) was added from day 0and kept in the medium until the end of the study. BMP7 (6 nM) was added2 days before day 0 and removed at day 0. Effects of differentiationmedium (MDM vs. EGM2), rosiglitazone (MDM+rosi vs. MDM) and BPM7(MDM+BMP7 vs. MDM) are shown as fold change (as compared to EGM2 cells)on UCP1 mRNA expression in human muscle-derived brown adipocyteprogenitor cells (CD31-).

The results are the mean±s.e.m. of arbitrary values normalized using thecorresponding cyclophilin A values. They are expressed in fold changevs. their respective non-induced (EGM2) values considered as 1 (n=3).

By contrast, in the CD31+ and CD56+ cell populations UCP1 mRNA wasundetectable in all but a few samples where it was at least 1,000-fold(10 cycles) lower in abundance than in the differentiated CD31− cells.

Example 12: Quantification of UCP1 mRNA by TaqMan Real-Time PCR

A robust method is provided herein, for detection of CD31− celldifferentiation into brown adipocytes by simultaneously quantifying mRNAspecies corresponding to the brown adipocyte marker UCP1 (FIG. 18A), theadipocyte marker FABP4 (FIG. 18B), and the “housekeeping” genecyclophilin A which was used as the internal control.

This method of the present invention permits analysis of a large numberof samples to identify agents that enhance the differentiation of CD31−cells into brown adipocytes. When differentiated into brown adipocytesCD31− cells express much higher levels of UCP1 and FABP4 mRNA for agiven level of cyclophilin A. UCP1 and FABP4 mRNA levels normalized tocyclophilin A mRNA levels give an indication of the level ofdifferentiation of the CD31− cells into brown adipocytes, independent ofthe total number of cells in the sample.

Quantification of UCP1, FABP4 and cyclophilin A mRNA by multiplexedTaqMan real-time PCR can thus be used to quantify differentiation of theCD31− cells into brown adipocytes.

Example 13: Quantification of UCP1 Protein by FluorescenceImmunohistochemistry (IHC)

Another robust method for detection of CD31− cell differentiation intobrown_adipocytes is provided herein, by quantifying UCP1 protein byfluorescence immunohistochemistry (IHC).

Using this method, UCP1 staining can be evaluated using automated highcontent imaging. GE Healthcare's InCell 1000 High Content Imagercombines automated microscopy with high content image analysis software(InCell Developer Toolbox). The reference compound rosiglitazone wasused to promote the differentiation of the CD31− cells and increase theexpression of UCP1. Using a primary antibody that recognizes UCP1, Abcamab23841, and a secondary antibody, Alexafluor 488 goat anti-rabbitantibody, we can quantify relative UCP1 levels per cell. With thismethod we found that rosiglitazone increased the level of UCP1 protein7-fold (UCP1 intensity per nucleus of 105,542 with rosiglitazone vs.14,815 without rosiglitazone) (FIG. 17A, 17B). It was also discoveredthat non-specific antibody background signal was very low, and thestandard deviation of mean UCP1 signal per cell was very small,indicating that the assay is suitable for high content screening.

Referring to FIGS. 17A-17B, culturing and differentiation of CD31− cellsinto brown adipocytes were performed as described in Methods without(FIG. 17A) or with (FIG. 17B) rosiglitazone (1 μM) added to theadipogenic medium (MDM) from day 0. Cells were differentiated for 15days, then fixed with 4% paraformaldehyde PBS pH 7.4, and incubated witha UCP1 antibody (Abcam ab23841). Alexafluor 488 goat anti-rabbitantibody was used to quantify relative UCP1 levels (green). Nuclei werestained with DAPI (blue). UCP1 signal intensity was measured using theGE Developer Toolbox software on images collected on the InCell 1000Automated High Content Imager. Numerical data was exported in tableformat and screenshots of how the algorithm processed the originalimages were also collected.

Fluorescence IHC detection of UCP1 can thus be used with a commerciallyavailable UCP1 antibody to quantify UCP1 protein and differentiation ofthe CD31− cells into brown adipocytes.

Materials and Methods

Except otherwise indicated, all organic and inorganic chemicals ofanalytical or molecular biology grade were purchased from Sigma ChemicalCo. (St Louis, Mich.) and Gibco BRL (New York, N.Y.). Rosiglitazone waspurchased from Cayman Chemical (#71742) and recombinant human BMP7(rhBMP7) was from R&D Systems (100 μg/ml, 6.3 μM, #354-BP-010).

Human Tissues

Human fetal tissues were obtained anonymously, following spontaneous,voluntary or therapeutic terminations of pregnancy, from Magee WomenHospital, University of Pittsburgh and Erasmus Medical Center,Rotterdam, The Netherlands, in compliance with the Institutional ReviewBoard protocol. Developmental age (16 to 24 or 18 to 19 weeks ofgestation) was estimated by measuring foot length. Informed consent tothe use of fetal tissues was obtained from the patients in allinstances. Adult human discarded abdominal subcutaneous WAT, originatingfrom 45-55 year old patients undergoing plastic surgery performed oneyear after gastric bypass, was kindly provided by Dr. Peter Rubin(Division of Plastic Surgery, University of Pittsburgh). The adultskeletal muscle tissue used for cell sorting was obtained post mortemfrom 50-78 year-old donors. The adult skeletal muscle used for the firstgroup of RT-PCR studies was obtained from the rectus abdominus duringsurgery for either lap banding, inguinal hernia or hysterectomy of 10lean male and female subjects. All subjects agreed to donate musclesamples during their operations and the protocol was approved by theMedical Ethical Review Committee of Deakin University. The average ageswere 45±3 years and the average body mass index was 22.2±0.8. The adultskeletal muscle used for the second group of RT-PCR studies was obtainedfrom the vastus lateralis of 7 obese type 2 diabetic male and femalepatients before and after 8 weeks of treatment with rosiglitazone (2×4mg per day). The average age was 63±4 years and the average body massindex was 29.9±3.8. The complete clinical profile of the patients hasbeen described in a previous publication [18]. All subjects agreed todonate muscle samples, and the protocol was approved by the MedicalEthical Review Committee of Maastricht University.

Adult Tissues Used for CD31− Cell Purification

Human skeletal muscle tissue samples from autopsy (25-80 year oldindividuals) were obtained from the National Disease ResearchInterchange (NDRI, Philadelphia, Pa.) and approved by NDRI's FeasibilityReview Committee (protocol #BOO1).

Mice

Animals were treated in accordance with the Centre Médical Universitaire(Genéve) institutional guidelines. They were housed individually andkept on a 12h light-dark cycle in a temperature-controlled room at 24°C. They were allowed ad libitum access to water and a standardlaboratory chow. The interscapular BAT of 4- to 6-week-old male 129Sv/ev mice were excised and their precursor cells isolated and culturedas previously described [19].

Immunohistochemistry

Fresh fetal and adult tissues were gradually frozen by immersion inisopentane cooled in liquid nitrogen. 5- to 7-μm sections were cut on acryostat (Microm HM 505 E), fixed with 50% acetone and 50% methanol,dried for 5 min at room temperature (RT), and then washed 3 times for 5min in phosphate-buffered saline. Non-specific binding sites wereblocked with 5% goat serum for 1 hour at RT. Sections were incubatedovernight at 4° C. with a CD34 mouse anti-human antibody (Serotech,1:50), then, after rinsing, for 1 hour at RT with a secondary goatanti-mouse biotinylated antibody (DAKO, 1:1000) and for 30 min at RTwith strep tavidin-Cy3 (Sigma, 1:1000) or for 2 hours at RT with aconjugated CD146-Alexa 488 mouse anti-human antibody (Chemicon, 1:200).Nuclei were stained with 4′,6-diamino-2-phenylindole dihydrochloride(Molecular Probes, 1:2000) for 5 min at RT. An isotype-matched negativecontrol was performed with each immunostaining.

Quantification of UCP1 Protein by Fluorescence Immunohistochemistry(IHC)

Culturing and differentiation of CD31− cells into brown adipocytes wereperformed using adipogenic differentiation medium lacking (MinimalDifferentiation Medium, MDM) or containing 1 μM rosiglitazone (ReferenceDifferentiation Medium, RDM). After 15 days of differentiation cellswere fixed with 4% Paraformaldehyde PBS pH 7.4, and incubated with aUCP1 antibody (Abcam ab23841) and Alexafluor 488 goat anti-rabbitantibody to quantify relative UCP1 levels (green) according to standardprotocols. Prior to fixation of cells, nuclei were labeled with 5 μMDAPI (blue) for 10 minutes. Each treatment condition was evaluated intriplicate in a 96-well plate corresponding to approximately 360-480cells for each data point in total. The InCell 1000 Developer Toolboxsoftware was used to develop an automated cell detection script tomeasure UCP1 signal intensity, using the nuclei and cytoplasm detectionalgorithms. As a readout, total intensity of UCP1 signal within the cellwas used, normalized to cell number.

Flow Cytometry

The vascular cells of fetal skeletal muscle, pancreas, lung and liver aswell as of adult muscle and WAT were analysed by flow cytometry. Freshfetal or adult muscle as well as fetal pancreas, lung and liver tissueswere cut into small pieces with a scalpel in Dulbecco's Modified EagleMedium high glucose (DMEM) containing 20% fetal bovine serum (FBS), 1%penicillin-streptomycin (PS) and collagenases IA-S, II-S and IV-S(Sigma,1 mg/mL), then incubated at 37° C. for 75 min (fetal tissues) or 90 min(adult tissues) with constant stirring. Final cell dissociation wasachieved between ground glass slides. Cells were washed withphosphate-buffered saline and centrifuged for 5 min at 350 g. They wereresuspended in DMEM, 20% FBS, filtered at 100 μm, stained with Trypanblue and counted after dead cell exclusion. The WAT stroma vascularfraction was prepared by collagenase digestion according to Champigny etal. [20].

Cells (10⁵ for analysis and around 30×10⁶ for sorting) were incubatedwith one of the following directly coupled mouse anti-human antibodies:CD45-APC Cy7 (Santa Cruz Biotechnologies, 1:200), CD45 PerCP Cy5.5(eBioscience #45-0459-73, 1:10), CD56-PE Cy7 (BD Pharmigen 1:100), CD56APC (BD Pharmigen #555518, 1:50), CD34-PE (DAKO, 1:100 or BD Pharmigen#555822, 1:20), CD146 unconjugated, BD Pharmigen: PE mouse anti-humanCD146 (BD Pharmigen #550315, 1:50), CD146-FITC (Serotec, 1:100), andanti-human CD31 (PECAM-1) FITC (eBioscience #11-0319-42, 1:10), each in1 ml DMEM, 20% FBS, 1% penicillin-streptomycin (PS), at 4° C. for 15min. After washing and centrifugation, cells were incubated with7-amino-actinomycin D (7-AAD, BD Pharmigen, 1:100) or the dye Hoechst33528 at 1 μg/ml (Molecular Probes-Invitrogen #H3569) for cellviability/dead cell exclusion, filtered at 70 μm and run on a FACS Ariaflow cytometer (Becton Dickinson). As negative controls, cell aliquotswere incubated with isotype-matched mouse IgGs conjugated to APC Cy7 (BDPharmigen, 1:100), PE Cy7 (BD Pharmigen, 1:100), PE (Chemicon, 1:100)and FITC (US Biological, 1:100) under the same conditions.

Cell Culture

Cells were seeded at 10,000 per cm² in 0.2% gelatin coated plates(24-well, BD Falcon #353047), cultured until confluency (2-4 days) at37° C. in Endothelial cell growth medium-2 (EGM2) (BulletKit growthmedium, Lonza #CC-3162) and until differentiation (10-16 more days).After 2 or 3 days in EGM2 medium the cells were induced to differentiateby replacing the medium with an adipogenic medium, which is amodification of the adipogenic medium described by Rodriguez et al. [21]and may or may not contain a differentiation inducing agent (e.g., PPARγagonist). The MDM described above contains: DMEM/Ham's F-12 50/50 Mix(3.151 g/1, 17.5 mM D-glucose, 3.651 g/1 L-glutamine) (Cellgro#10-090-CV), 5 μg/ml (0.86 μM) insulin, 10 μg/ml transferrin, 0.2 nM3,3′,5-triiodo-L-thyronine, 100 μM 3-isobutyl-1-methylxanthine, 1 μMdexamethasone, and 1% penicillin-streptomycin. If rosiglitazone is usedas a differentiation inducing agent, it can be supplied at 1 μM or anyother concentration sufficient to induce differentiation of BATprogenitor cells into adipocytes.

For cell expansion studies, confluent cells grown in EGM2 medium onlywere detached by treatment with trypsin-EDTA for 3-5 min at 37° C., andthen split 1:3 or 1:4 and cultured as described above. Human whiteadipocytes in primary culture used in the oximetry studies were obtainedas previously described [22].

RT-PCR

Total cell RNA was prepared using the kit NucleoSpin® RNAII (Clontech,Palo Alto, Calif.), Extract-all solution (Eurobio, Courtaboeuf, France)or PureLink RNA Isolation Kit (Invitrogen #12183-016) and quantified byBiophotometry (Biophotometer, Eppendorf). Oligo-dT primed First strandcDNA were synthesized using the Superscript™ II RNase H ReverseTranscription kit (Invitrogen, Carlsbad, Calif.) and oligo-dT primers orthe High Capacity cDNA Reverse Transcription kit (Applied Biosystems,Foster City, Calif.) and random primers.

Quantitative real-time PCR was performed using ABI rapid thermal cyclersystem and a SYBR Green PCR master mix (Applied Biosystems, Foster City,Calif.), or an ABI 7300 Real-time PCR instrument, a TaqMan GeneExpression Master Mix (Applied Biosystems #4369016) and TaqMan GeneExpression Pre-formulated Assays for human uncoupling protein-1 (GenBankNM_021833) (Applied Biosystems Assay ID Hs01084772_m1, TaqMan GeneExpression Assay part #4351372) and for human peptidylprolyl isomerase A“cyclophilin A” (GenBank NM_021130) (Applied Biosystems Assay IDHs99999904 ml, TaqMan® Gene Expression Assay part #4448485). CyclophilinA was used as a control to account for any variations due to theefficiency of the reverse transcription. The upstream and downstreamoligonucleotide primers were chosen on both sides of an intron toprevent amplification of contaminating genomic DNA.

The primers used for real time quantitative PCR in human cells and inmouse brown adipocytes are as follows:

hUCP1 (SEQ ID NO: 1) Sense primer: 5′-CCTCACCGCAGGGAAAGAA-3′(SEQ ID NO: 2) Antisense primer: 5′-CTAACGACTGGAGGAGTGGCA-3′Amplicon position: 429-504 Accession No.: NM_021833 mUCP1 (SEQ ID NO: 3)Sense primer: 5′-CGATGTCCATGTACACCAAGGA-3′ (SEQ ID NO: 4)Antisense primer: 5′-TTGTGGCTTCTnTCTGCGA-3′ Amplicon position: 996-1063Accession No.: NM_009463.2 hleptin (SEQ ID NO: 5) Sense primer:5′-CCAAAACCCTCATCAAGACAATT-3′ (SEQ ID NO: 6) Antisense primer:5′-AAGTCACCGGTTTGGACTTCA-3′ Amplicon position: 143-238 Accession No.:BC069323 hcyclophilin A (SEQ ID NO: 7) Sense primer:5′-CATCTGCACTGCCAAGACTGA-3′ (SEQ ID NO: 8) Antisense primer:5′-GCAAAGTGAAAGAAGGCATGAA-3′ Amplicon position: 466-537 Accession No.:NM_203431 mcyclophilin A (SEQ ID NO: 9) Sense primer:S′-CAAATGCTGGACCAAACACAA-3′ (SEQ ID NO: 10) Antisense primer:5′-CCATCCAGCCATTCAGTCTTG-3′ Amplicon position: 343-412 Accession No.:NM_008907

Primers used for real time quantitative PCR in human skeletal muscle areas follows:

hUCP1 (SEQ ID NO: 11) Sense primer: S′-TCCGGCTCCAGGTCCAA-3′(SEQ ID NO: 12) Antisense primer: 5′-TGATTGTTCCCAGGACACCTTT-3′Amplicon position: 240-311 Accession No.: NM_021833 hcyclophilin A(SEQ ID NO: 7) Sense primer: 5′-CATCTGCACTGCCAAGACTGA-3′ (SEQ ID NO: 8)Antisense primer: 5′-GCAAAGTGAAAGAAGGCATGAA-3′ Amplicon position:466-537 Accession No.: NM_203431

Sequences and primers used for analytical PCR are as follows:

CD31 Accession No.: NM_000442 CD34 (SEQ ID NO: 13) Sense primer:5′-CATCACTGGCTATTTCCTGATG-3′ (SEQ ID NO: 14) Antisense primer:′5-AGCCGAATGTGTAAAGGACAG-3′ Amplicon position: 1172-1591 Accession No.:M81104 CD56 (SEQ ID NO: 15) Sense primer: 5′-GTATTTGCCTATCCCAGTGCC-3′(SEQ ID NO: 16) Antisense primer: 5′-CATACTTCTTCACCCACTGCTC-3′Amplicon position: 542-873 Accession No.: BC014205 CD45 (SEQ ID NO: 17)Sense primer: 5′-CATGTACTGCTCCTGATAAGAC-3′ (SEQ ID NO: 18)Antisense primer: 5′-GCCTACACTTGACATGCATAC-3′ Amplicon position:940-1579 Accession No.: Y00638 CD146 (SEQ ID NO: 19) Sense primer:5′-AAGGCAACCTCAGCCATGTCG-3′ (SEQ ID NO: 20) Antisense primer:5′-CTCGACTCCACAGTCTGGGAC-3′ Amplicon position: 168-603 Accession No.:M28882 β-actin (SEQ ID NO: 21) Sense primer: 5′-CCTCGCCTTTGCCGATCC-3′(SEQ ID NO: 22) Antisense primer: 5′-GGAATCCTTCTGACCCATGC-3′Amplicon position: 25-229 Accession No.: NM_001101.

Arbitrary units were determined by normalizing target mRNA levels tocyclophilin mRNA levels (based on Cts), wherein the cyclophilin levelswere first divided by 100,000 for ease of reference. For example, aratio of target mRNA to cyclophilin mRNA of 0.01797 is expressed as1797.

Alternatively, quantitative real-time PCR was performed using an AppliedBiosystems 7300 real-time PCR instrument (with SDS Software v1.4.0.25),TaqMan Gene Expression Master Mix (Applied Biosystems #4369016), andTaqMan Gene Expression Pre-formulated Assays for human uncouplingprotein-1 “UCP1” (GenBank NM_021833) [FAM MGB probe] (Applied BiosystemsAssay ID Hs00222453_m1, TaqMan Gene Expression Assay part #4351370) andfor human peptidylprolyl isomerase A “cyclophilin A” (GenBank NM_021130)[VIC MGB probe] (Applied Biosystems Assay ID Hs99999904_m1, TaqMan GeneExpression Assay part #4448485). Custom TaqMan Gene Expression reagentswere developed for simultaneous measurement of human fatty acid bindingprotein 4 “FABP4” (GenBank NM_001442) in a multiplexed fashion (withUCP1 and cyclophilin A): TaqMan MGB Probe [NED]: CAG GAA AGT CAA GAG CACCA (Applied Biosystems #4316034; SEQ ID NO:30), Sense primer: TCA TACTGG GCC AGG AAT TT (Applied Biosystems #4304971; SEQ ID NO:31) andAntisense primer: TGC ACA TGT ACC AGG ACA CC (Applied Biosystems#4304971; SEQ ID NO:32).

Cyclophilin A was used as a control to account for any variations due tothe efficiency of reverse transcription. Arbitrary units were determinedby normalizing target mRNA levels to cyclophilin A mRNA levels (based onCts).

Validation of the Human UCP1 Amplicon

The PCR-amplified fragment was cloned into the pCR2.1-TOPO vectorthrough the TOPO-TA cloning system (Invitrogen, Carlsbad, Calif.) andpurification of color-selected colonies was performed using the QiaprepSpin Miniprep (Qiagen, Hilden, Germany). Sequences were determined witholigonucleotide M 13 Reverse on the pCR2.1-TOPO vector using the AppliedBiosystem Big Dye sequencing kit on an ABI 3700 automated sequencer(Applied Biosystems, Foster City, Calif.).

Western Blots

Cultured cells were collected with a rubber policeman in 200 μl of RIPAbuffer (150 mM NaCl, 1% Nonidet P-40, 0.5% Na deoxycholate, 0.1% SDS,1:200 protease inhibitor cocktail (Sigma Chemical Co, St Louis, Mich.)and 50 mM Tris/HCl pH 8.0). Human BAT and skeletal muscle werehomogenized in the above RIPA buffer. The protein content was determinedaccording to the technique of Lowry [23]. Western blots were performedas previously described [24]. The UCP1 protein was detected using a1/500 diluted rabbit anti-mouse UCP1 polyclonal primary antibodygenerously provided by Dr. B. Cannon (Stockholm, Sweden). This antibodyhad been raised against the C-terminal decapeptide of mouse UCP1, thatshares 80% identity with human UCP1 and 0 and 10% identities with humanUCP2 and UCP3, respectively. Glyceraldehyde phosphate dehydrogenase(GAPDH) protein was detected using a 1/5000 diluted mouse anti-mouseGAPDH monoclonal primary antibody (Chemicon International, Inc,Temecula, Calif.). 1/5000 diluted goat anti-rabbit or anti-mouseperoxidase-labelled secondary antibodies (Sigma-Aldrich, St. Louis, Mo.or Bio-Rad, Hercules, Calif.) were used. A SeeBlue® Plus 2 Pre-stainedStandard Ladder (Invitrogen, Carlsbad, Calif.) was used. Protein signalswere detected by chemiluminescence using a standard ECL kit anddeveloped on a Hyperfilm ECL film.

High-Resolution O₂ Consumption Measurement

Oxygen consumption was measured using a 2-injection chambersrespirometer equipped with a Peltier thermostat, Clark-type electrodes,and integrated electromagnetic stirrers (Oroboros® Oxygraph, Oroboros,Innsbruck, Austria). Measurements were performed at 37° C. withcontinuous stirring in 2 ml of DMEM F12, 10% new born calf serum. Underthese conditions, the serum provided the fatty acids necessary tosustain UCP1 uncoupling activity. Before each 02 consumptionmeasurement, the medium in the chambers was equilibrated with air for 30min, and freshly trypsinized cells were transferred into therespirometer glass chambers. After observing steady-state respiratoryflux, ATP synthase was inhibited with oligomycin (0.25-0.5 mg/1) andcells were titrated with the uncoupler carbonyl cyanide3-chloro-phenylhydrazone up to optimum concentrations in the range of1-2 μM. The respiratory chain was inhibited by antimycin A (1 μg/ml).Oxygen consumption was calculated using DataGraph software (Oroborossoftware).

CD31− Cell Respiration Measurement

Respiration rate of CD31− cells before differentiation and upondifferentiation into brown adipocytes was measured using a SeahorseBioscience XF24 Extracellular Flux Analyzer (Seahorse Bioscience,Billerica, Mass.). Adult-derived CD31− cells were first cultured inSeahorse plates in either EGM2 (i.e., undifferentiated progenitors) orMDM and rosiglitazone (1 μM)+BMP7 (6 nM) for 3 days (fullydifferentiated brown adipocytes). Next, measurements of respiration weretaken at baseline, in the presence of oligomycin (5 μM) (so-called“State 4” respiration, or proton leak) and in the presence of FCCP(titration: 0.5 μM, 1 μM and 5 μM) (so-called “State 3u” respiration, ormaximally uncoupled).

Gene Chip Analysis

The total RNA of fetal muscle CD34+ cells expanded in culture for up to3 passages (4 weeks) and of human muscle biopsies were prepared asdescribed above. The quality assurance measurements, the preparation ofthe cRNA targets and the gene chip analyses using Illumina Human WG-6BeadChip were performed by Expression Analysis, Inc. (Durham, N.C.).BeadStudio nonparametric methods were used for the computation ofDetection P-Values.

Statistical Analysis

Data are expressed as means±s.e.m. Significances were evaluated usingthe unpaired Student's t-test. A paired Student's t-test was used todetermine the effects of rosiglitazone on human skeletal muscle UCP1mRNA levels. Significances were set at p<0.05.

Cloning of the Human UCP1 Promoter/Enhancer Region

To develop a screening strategy, the human UCP1 promoter/enhancer wassubcloned as follows.

A human BAC (bacterial artificial chromosome) clone #RP11-5K16 (AC108019) containing the human UCP1 (uncoupling protein-1)promoter/enhancer region was obtained from the CHORI (Children'sHospital Oakland Research Institute) BAC-PAC resources service. Theselected promoter/enhancer region starts at position −25 upstream of the5′ UTR (UnTranslated Region) of the human UCP1 gene (accession number:NM_021833). Based on the human UCP1 gene initiation codon, the fullcloned promoter/enhancer sequence locates between position −149 and−6269.

Primer sets were designed to amplify either:

(i) the full targeted promoter/enhancer region (6120 bp starting atposition −25 upstream of the UCP1 5′ UTR):

Left primer: (SEQ ID NO: 23)5′-TCGTAAGCTTAGAGGCGGCGGCTGCAGACGGAGCGCGGTGTT-3′ Right primer:(SEQ ID NO: 24) 5′-ACGAAGATCTCATTACCCCAAATAGCATCACA-3′(ii) the proximal targeted promoter/enhancer region (3685 bp upstream ofthe −25 nucleotide of the UCP1 5′ UTR):

Left primer: (SEQ ID NO: 25)5′-TCGTAAGCTTAGAGGCGGCGGCTGCAGACGGAGCGCGGTGTT-3′ Right primer:(SEQ ID NO: 26) 5′-ACGAACCGGTCAGAAGTGGTGAAGCCAGCCTGC-3′(iii) the distal targeted promoter/enhancer region (2435 bp upstream ofthe proximal targeted promoter/enhancer region) as indicated:

Left primer: (SEQ ID NO: 27) 5′-TCGTACCGGTACAGGCTCTGGGAAGTAGGAGAAAGT-3′Right primer: (SEQ ID NO: 28) 5′-ACGAAGATCTCATTACCCCAAATAGCATCACA-3′

Each primer contains a restriction site to facilitate subsequent cloningin mammalian expression vector (see below).

Cloning of the promoter/enhancer in PCR reaction was performed with 500ng of BAC #RP11-5K16 as template, using Takara Ex Taq DNA Polymerase kit(Clontech) for amplification. PCR program steps were as follow:Initialization step: 92° C. for 2 minutes; followed by 28 cycles of:[denaturation: 92° C., 30 seconds; annealing: 59° C., 40 seconds;extension: 68° C., 5 minutes 30 second]; with a final elongation step:68° C., 8 minutes.

The full promoter/enhancer, proximal or distal promoter/enhancer weresubsequently subcloned in the reporter/MAR element-containing vectorpl_68_GFP at the BglII/HindIII sites, replacing the SV40 promotercassette [25]. Alternatively, the luciferase-based pGL3 Basic vector(Promega) was also used as another reporter type, using the sameBglII/NcoI sites for subcloning purpose.

The human UCP1 promoter sequence cloned was confirmed bystate-of-the-art sequencing, performed at biotechnology company, FastensSA, Switzerland. The sequence of the human UCP1 promoter sequence isprovided in the Sequence Listing as SEQ ID NO:29, wherein the entireSequence Listing is incorporated by reference in its entirety.

Pictures for Cell Morphology

Pictures of cells were taken using a hand-held digital camera (NikonCoolpix 950) and inverted microscope (Nikon TMS) used for cell cultureobservations; images were optimized using Adobe Photoshop Elements 8functions for Auto Contrast and Auto Levels.

The section headings and subheadings used in this specification are fororganizational purposes only and are not to be construed as limiting thesubject matter described in any way. Further, while the presentteachings are described in conjunction with various embodiments, it isnot intended that the present teachings be limited to such embodiments.On the contrary, the present teachings encompass various alternatives,modifications, and equivalents as will be appreciated by those of skillin the art.

Other Embodiments

Various aspects of the present invention may be used alone, incombination, or in a variety of arrangements not specifically discussedin the embodiments described in the foregoing and is therefore notlimited in its application to the details and arrangement of componentsset forth in the foregoing description or illustrated in the drawings.For example, aspects described in one embodiment may be combined in anymanner with aspects described in other embodiments.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

Also, the phraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting. The use of“including,” “comprising,” or “having,” “containing,” “involving,” andvariations thereof herein, is meant to encompass the items listedthereafter and equivalents thereof as well as additional items.

The present invention provides among other things novel methods andcompositions for BAT progenitor cells. While specific embodiments of thesubject invention have been discussed, the above specification isillustrative and not restrictive. Many variations of the invention willbecome apparent to those skilled in the art upon review of thisspecification. The full scope of the invention should be determined byreference to the claims, along with their full scope of equivalents, andthe specification, along with such variations.

INCORPORATION BY REFERENCE

All publications, patents and patent applications referenced in thisspecification are incorporated herein by reference in their entirety forall purposes to the same extent as if each individual publication,patent or patent application were specifically indicated to be soincorporated by reference.

The Sequence Listing filed as an ASCII text file via EFS-Web (file name:“130204_010202_SEQListing”; date of creation: Mar. 27, 2015; size:14,155 bytes) is hereby incorporated by reference in its entirety.

REFERENCES

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We claim:
 1. A brown adipose tissue (BAT) progenitor cell, wherein theBAT progenitor cell is isolated from human skeletal muscle and capableof differentiating into brown adipocyte, wherein the BAT progenitor cellexpresses a first cell surface marker associated with endothelial cells,said first cell surface marker being detectable in an antibody basedassay using a first antibody; and wherein the BAT progenitor cell issubstantially free of a second cell surface marker associated withendothelial cells, said second cell surface marker being substantiallyundetectable in said antibody based assay using a second antibody. 2.The BAT progenitor cell of claim 1, further comprising at least one of:wherein the BAT progenitor cell is substantially free of a third cellsurface marker associated with hematopoietic cells, said third cellsurface marker being substantially undetectable in said antibody basedassay using a third antibody; wherein the BAT progenitor cell issubstantially free of a fourth cell surface marker associated withmyogenic cells, said fourth cell surface marker being substantiallyundetectable in said antibody based assay using a fourth antibody;and/or wherein the BAT progenitor cell is substantially free of a fifthcell surface marker associated with pericytes, said fifth cell surfacemarker being substantially undetectable in said antibody based assayusing a fifth antibody.
 3. The BAT progenitor cell of claim 1, whereinthe first cell surface marker is CD34 and the first antibody is ananti-CD34 antibody.
 4. The BAT progenitor cell of claim 1, wherein thesecond cell surface marker is CD31 and the second antibody is ananti-CD31 antibody.
 5. The BAT progenitor cell of claim 2, wherein thethird cell surface marker is CD45 and the third antibody is an anti-CD45antibody.
 6. The BAT progenitor cell of claim 2, wherein the fourth cellsurface marker is CD56 and the fourth antibody is an anti-CD56 antibody.7. The BAT progenitor cell of claim 2, wherein the fifth cell surfacemarker is CD146 and the fifth antibody is an anti-CD146 antibody.
 8. TheBAT progenitor cell of claim 1, wherein the BAT progenitor cellproliferates in a proliferation medium.
 9. The BAT progenitor cell ofclaim 8, wherein the proliferation medium comprises bone morphogenicprotein-7 (BMP7).
 10. The BAT progenitor cell of claim 1, wherein theBAT progenitor cell differentiates into a brown adipocyte in adifferentiation medium.
 11. The BAT progenitor cell of claim 10, whereinthe differentiation medium is Minimal Differentiation Medium (MDM). 12.The BAT progenitor cell of claim 10, wherein the differentiation mediumcomprises a peroxisome proliferator-activated receptor gamma (PPARγ)agonist.
 13. A population of cells isolated from skeletal muscle,comprising at least one BAT progenitor cell of claim
 1. 14. A method foridentifying an agent that induces expression or activity levels of UCP1,comprising: providing the BAT progenitor cell of claim 1; contacting theBAT progenitor cell with an agent; and determining if the BAT progenitorcell exhibits an increase in UCP1 expression or activity.
 15. Adifferentiation medium comprising at least one BAT progenitor cell ofclaim
 1. 16. The differentiation medium of claim 15, wherein thedifferentiation medium is Minimal Differentiation Medium (MDM).
 17. Thedifferentiation medium of claim 15, wherein the differentiation mediumcomprises a peroxisome proliferator-activated receptor gamma (PPARγ)agonist and/or bone morphogenic protein-7 (BMP7).